Materials and Methods for Treatment of Myotonic Dystrophy Type 1 (DM) and Other Related Disorders

ABSTRACT

The present application provides materials and methods for treating a patient with one or more conditions associated with DMPK whether ex vivo or in vivo. In addition, the present application provides materials and methods for editing and/or modulating the expression of DMPK gene in a cell by genome editing.

FIELD

The present disclosure relates to the field of gene editing andspecifically to the alteration of the Dystrophia Myotonica-ProteinKinase (DMPK) gene.

RELATED APPLICATIONS

This application is a continuation of U.S. Application No. 16/312,651,filed Dec. 21, 2018, which is a 371 U.S. National Phase Application ofPCT/IB2017/053816, filed Jun. 27, 2017, which claims the benefit of U.S.Provisional Application No. 62/355,949 filed Jun. 29, 2016 and U.S.Provisional Application No. 62/461,875 filed Feb. 22, 2017, all of whichare incorporated herein in their entirety by reference.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form(filename: 2022-08-11_01245-0022-01US-ST25, 14,832,682 bytes - ASCIItext file; created Aug. 11, 2022), which is incorporated herein byreference in its entirety and forms part of the disclosure.

BACKGROUND

Genome engineering refers to the strategies and techniques for thetargeted, specific modification of the genetic information (genome) ofliving organisms. Genome engineering is a very active field of researchbecause of the wide range of possible applications, particularly in theareas of human health. For example, genome engineering can be used toalter (e.g., correct or knock-out) a gene carrying a harmful mutation,or to explore the function of a gene. Early technologies developed toinsert a transgene into a living cell were often limited by the randomnature of the insertion of the new sequence into the genome. Randominsertions into the genome may result in disrupting normal regulation ofneighboring genes leading to severe unwanted effects. Furthermore,random integration technologies offer little reproducibility, as thereis no guarantee that the sequence would be inserted at the same place intwo different cells. Recent genome engineering strategies, such as zincfinger nucleases (ZFNs), transcription activator like effector nucleases(TALENs), homing endonucleases (HEs) and MegaTALs, enable a specificarea of the DNA to be modified, thereby increasing the precision of thealteration compared to early technologies. These newer platforms offer amuch larger degree of reproducibility, but still have their limitations.

Despite efforts from researchers and medical professionals worldwide whohave been trying to address genetic disorders, and despite the promiseof genome engineering approaches, there still remains a critical needfor developing safe and effective treatments involving DMPK relatedindications.

By using genome engineering tools to create permanent changes to thegenome that can address the DMPK related disorders or conditions with asfew as a single treatment, the resulting therapy may completely remedycertain DMPK related indications and/or diseases.

SUMMARY

Provided herein are cellular, ex vivo and in vivo methods for creatingpermanent changes to the genome by deleting and/or correcting thetrinucleotide repeat expansion or replacing one or more nucleotidebases, or one or more exons and/or introns within or near the DystrophiaMyotonica-Protein Kinase (DMPK) gene, or otherwise introducinginsertions, deletions or mutations of at least one nucleotide within ornear the DMPK gene or other DNA sequences that encode regulatoryelements of the DMPK gene, by genome editing. Such methods can restorethe Dystrophia Myotonica-Protein Kinase (DMPK) protein activity and/orreduce or eliminate the expression or function of aberrant DMPK geneproducts, which can be used to treat a DMPK related condition ordisorder such as Myotonic Dystrophy Type 1. Also provided herein arecomponents and compositions, and vectors for performing such methods.

Provided herein is a method for editing a Dystrophia Myotonica-ProteinKinase (DMPK) gene in a cell by genome editing comprising: introducinginto the cell one or more deoxyribonucleic acid (DNA) endonucleases toeffect one or more single-strand breaks (SSBs) or double-strand breaks(DSBs) within or near the DMPK gene that results in permanent deletionof the expanded trinucleotide repeat or replacement of one or morenucleotide bases, or one or more exons and/or introns within or near theDMPK gene, thereby restoring the DMPK gene function.

Also provided herein is a method for editing a DystrophiaMyotonica-Protein Kinase (DMPK) gene in a cell by genome editingcomprising: introducing into the cell one or more deoxyribonucleic acid(DNA) endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the DMPK gene or DMPKregulatory elements that results in one or more permanent insertion,deletion or mutation of at least one nucleotide within or near the DMPKgene, thereby reducing or eliminating the expression or function ofaberrant DMPK gene products.

Also provided herein is an ex vivo method for treating a patient havinga DMPK related condition or disorder comprising: isolating a muscle cellor muscle precursor cell from a patient; editing within or near aDystrophia Myotonica-Protein Kinase (DMPK) gene or other DNA sequencesthat encode regulatory elements of the DMPK gene of the muscle cell ormuscle precursor cell; and implanting the genome-edited muscle cell ormuscle precursor cell into the patient.

In some aspects, the editing step comprises: introducing into the musclecell or muscle precursor cell one or more deoxyribonucleic acid (DNA)endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the DMPK gene that results inpermanent deletion of the expanded trinucleotide repeat or replacementof one or more nucleotide bases, or one or more exons and/or intronswithin or near the DMPK gene, thereby restoring the DMPK gene function.

In some aspects, the editing step comprises: introducing into the musclecell or muscle precursor cell one or more deoxyribonucleic acid (DNA)endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the DMPK gene or DMPKregulatory elements that results in one or more permanent insertion,deletion or mutation of at least one nucleotide within or near the DMPKgene, thereby reducing or eliminating the expression or function ofaberrant DMPK gene products. In some aspects, the muscle cell is askeletal muscle cell. In some aspects, the muscle cell is a smoothmuscle cell. In some aspects, the muscle cell is a cardiac muscle cell.

Also provided herein is an ex vivo method for treating a patient havinga DMPK related condition or disorder comprising: creating a patientspecific induced pluripotent stem cell (iPSC); editing within or near aDystrophia Myotonica-Protein Kinase (DMPK) gene or other DNA sequencesthat encode regulatory elements of the DMPK gene of the iPSC;differentiating the genome-edited iPSC into a skeletal muscle cell, asmooth muscle cell, a cardiac muscle cell or a Pax7+ myocyte progenitorcell; and implanting the skeletal muscle cell, smooth muscle cell,cardiac muscle cell or Pax7+ myocyte progenitor cell into the patient.

In some aspects, the editing step comprises: introducing into the iPSCone or more deoxyribonucleic acid (DNA) endonucleases to effect one ormore single-strand breaks (SSBs) or double-strand breaks (DSBs) withinor near the DMPK gene that results in permanent deletion of the expandedtrinucleotide repeat or replacement of one or more nucleotide bases, orone or more exons and/or introns within or near the DMPK gene, therebyrestoring the DMPK gene function.

In some aspects, the editing step comprises: introducing into the iPSCone or more deoxyribonucleic acid (DNA) endonucleases to effect one ormore single-strand breaks (SSBs) or double-strand breaks (DSBs) withinor near the DMPK gene or DMPK regulatory elements that results in one ormore permanent insertion, deletion or mutation of at least onenucleotide within or near the DMPK gene, thereby reducing or eliminatingthe expression or function of aberrant DMPK gene products.

Also provided herein is an ex vivo method for treating a patient withhaving a DMPK related condition or disorder comprising: isolating amesenchymal stem cell from the patient; editing within or near aDystrophia Myotonica-Protein Kinase (DMPK) gene or other DNA sequencesthat encode regulatory elements of the DMPK gene of the mesenchymal stemcell; differentiating the genome-edited mesenchymal stem cell into askeletal muscle cell, a smooth muscle cell, a cardiac muscle cell or aPax7+ myocyte progenitor cell; and implanting the skeletal muscle cell,smooth muscle cell, cardiac muscle cell or Pax7+ myocyte progenitor cellinto the patient.

In some aspects, the editing step comprises: introducing into themesenchymal stem cell one or more deoxyribonucleic acid (DNA)endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the DMPK gene that results inpermanent deletion of the expanded trinucleotide repeat or replacementof one or more nucleotide bases, or one or more exons and/or intronswithin or near the DMPK gene, thereby restoring the DMPK gene function.

In some aspects, the editing step comprises: introducing into themesenchymal stem cell one or more deoxyribonucleic acid (DNA)endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the DMPK gene or DMPKregulatory elements that results in one or more permanent insertion,deletion or mutation of at least one nucleotide within or near the DMPKgene, thereby reducing or eliminating the expression or function ofaberrant DMPK gene products.

Also provided herein is an in vivo method for treating a patient with aDMPK related disorder comprising: editing the DystrophiaMyotonica-Protein Kinase (DMPK) gene in a cell of the patient.

In some aspects, the editing step comprises: introducing into the cellone or more deoxyribonucleic acid (DNA) endonucleases to effect one ormore single-strand breaks (SSBs) or double-strand breaks (DSBs) withinor near the DMPK gene that results in permanent deletion of the expandedtrinucleotide repeat or replacement of one or more nucleotide bases, orone or more exons and/or introns within or near the DMPK gene, therebyrestoring the DMPK gene function.

In some aspects, the editing step comprises: introducing into the cellone or more deoxyribonucleic acid (DNA) endonucleases to effect one ormore single-strand breaks (SSBs) or double-strand breaks (DSBs) withinor near the DMPK gene or DMPK regulatory elements that results in one ormore permanent insertion, deletion or mutation of at least onenucleotide within or near the DMPK gene, thereby reducing or eliminatingthe expression or function of aberrant DMPK gene products.

In some aspects, the cell is a muscle cell or muscle precursor cell. Insome aspects, the muscle cell is a skeletal muscle cell. In someaspects, the muscle cell is a smooth muscle cell. In some aspects,muscle cell is a cardiac muscle cell. In some aspects, the one or moredeoxyribonucleic acid (DNA) endonuclease is delivered to the muscle cellor muscle precursor cell by local injection into the desired muscle.

Also provided herein is a method of altering the contiguous genomicsequence of a DMPK gene in a cell comprising: contacting the cell withone or more deoxyribonucleic acid (DNA) endonuclease to effect one ormore single-strand breaks (SSBs) or double-strand breaks (DSBs). In someaspects, the alteration of the contiguous genomic sequence occurs in oneor more exons of the DMPK gene. In some aspects, the alteration of thecontiguous genomic sequence occurs in the 3’ untranslated region (UTR)of the DMPK gene.

In some aspects, the one or more deoxyribonucleic acid (DNA)endonuclease is selected from any of those sequences in SEQ ID NOs:1-620 and variants having at least 90% homology to any of the sequenceslisted in SEQ ID NOs: 1-620.

In some aspects, the one or more deoxyribonucleic acid (DNA)endonuclease is one or more protein or polypeptide. In some aspects, theone or more deoxyribonucleic acid (DNA) endonuclease is one or morepolynucleotide encoding the one or more DNA endonuclease. In someaspects, the one or more deoxyribonucleic acid (DNA) endonuclease is oneor more ribonucleic acid (RNA) encoding the one or more DNAendonuclease. In some aspects, the one or more ribonucleic acid (RNA) isone or more chemically modified RNA. In some aspects, the one or moreribonucleic acid (RNA) is chemically modified in the coding region. Insome aspects, the one or more polynucleotide or one or more ribonucleicacid (RNA) is codon optimized.

In some aspects, the method further comprises introducing one or moregRNA or one or more sgRNA. In some aspects, the one or more gRNA or oneor more sgRNA comprises a spacer sequence that is complementary to asequence within or near the expanded trinucleotide repeat in the DMPKgene. In some aspects, the one or more gRNA or one or more sgRNAcomprises a spacer sequence that is complementary to a DNA sequencewithin or near the DMPK gene. In some aspects, the one or more gRNA orone or more sgRNA comprises a spacer sequence that is complementary to asequence flanking the DMPK gene or other sequence that encodes aregulatory element of the DMPK gene. In some aspects, the one or moregRNA or one or more sgRNA is chemically modified.

In some aspects, the one or more gRNA or one or more sgRNA ispre-complexed with the one or more deoxyribonucleic acid (DNA)endonuclease. In some aspects, the pre-complexing involves a covalentattachment of the one or more gRNA or one or more sgRNA to the one ormore deoxyribonucleic acid (DNA) endonuclease.

In some aspects, the one or more deoxyribonucleic acid (DNA)endonuclease is formulated in a liposome or lipid nanoparticle. In someaspects, the one or more deoxyribonucleic acid (DNA) endonuclease isformulated in a liposome or a lipid nanoparticle which also comprisesthe one or more gRNA or one or more sgRNA.

In some aspects, the one or more deoxyribonucleic acid (DNA)endonuclease is encoded in an AAV vector particle. In some aspects, theone or more gRNA or one or more sgRNA is encoded in an AAV vectorparticle. In some aspects, the one or more deoxyribonucleic acid (DNA)endonuclease is encoded in an AAV vector particle which also encodes theone or more gRNA or one or more sgRNA. In some aspects, the AAV vectorparticle is selected from the group consisting of any of those listed inSEQ ID NOs: 4734-5302 and Table 2.

In some aspects, the method further comprises introducing into the cella donor template comprising at least a portion of the wild-type DMPKgene. In some aspects, at least a portion of the wild-type DMPK genecomprises one or more sequences selected from a group consisting of: aDMPK exon, a DMPK intron, and a sequence comprising an exon:intronjunction of DMPK. In some aspects, the donor template compriseshomologous arms to the genomic locus of the DMPK gene. In some aspects,the donor template is either a single or double stranded polynucleotide.

In some aspects, the donor template is encoded in an AAV vectorparticle. In some aspects, the AAV vector particle is selected from thegroup consisting of any of those disclosed in SEQ ID NOs: 4734-5302 andTable 2. In some aspects, the one or more polynucleotide encoding one ormore deoxyribonucleic acid (DNA) endonuclease is formulated into a lipidnanoparticle, and the one or more gRNA or one or more sgRNA is deliveredto the cell ex vivo by electroporation and the donor template isdelivered to the cell by an adeno-associated virus (AAV) vector. In someaspects, the one or more polynucleotide encoding one or moredeoxyribonucleic acid (DNA) endonuclease is formulated into a liposomeor lipid nanoparticle which also comprises the one or more gRNA or oneor more sgRNA and the donor template.

Also provided herein is a single-molecule guide RNA comprising: at leasta spacer sequence that is an RNA sequence selected from any of SEQ IDNOs: 5305-20697. In some aspects, the single-molecule guide RNA furthercomprises a spacer extension region. In some aspects, thesingle-molecule guide RNA further comprises a tracrRNA extension region.In some aspects, the single-molecule guide RNA is chemically modified.

In some aspects, the single-molecule gudie RNA is pre-complexed with aDNA endonuclease. In some aspects, the DNA endonuclease is a Cas9 orCPfl endonuclease. In some aspects, the Cas9 or Cpfl endonuclease isselected from a group consisting of: S. pyogenes Cas9, S. aureus Cas9,N. meningitides Cas9, S. thermophilus CRISPR1 Cas9, S. thermophilusCRISPR 3 Cas9, T. denticola Cas9, L. bacterium ND2006 Cpfl andAcidaminococcus sp. BV3L6 Cpfl, and variants having at least 90%homology to these endonucleases. In some aspects, the Cas9 or Cpflendonuclease comprises one or more nuclear localization signals (NLSs).In some aspects, at least one NLS is at or within 50 amino acids of theamino-terminus of the Cas9 or Cpfl endonuclease and/or at least one NLSis at or within 50 amino acids of the carboxy-terminus of the Cas9 orCpfl endonuclease.

Also provided herein is a non-naturally occurring CRISPR/Cas systemcomprising a polynucleotide encoding a Cas9 or Cpfl endonuclease and atleast one single-molecule guide RNA described herein. In some aspects,the polynucleotide of the CRISPR/Cas system described herein andencoding a Cas9 or Cpfl endonuclease is selected from the groupconsisting of: S. pyogenes Cas9, S. aureus Cas9, N. meningitides Cas9,S. thermophilus CRISPR1 Cas9, S. thermophilus CRISPR 3 Cas9, T.denticola Cas9, L. bacterium ND2006 Cpfl and Acidaminococcus sp. BV3L6Cpfl, and variants having at least 90% homology to the endonucleases. Insome aspects, the polynucleotide encoding a Cas9 or Cpfl endonucleasecomprises one or more nuclear localization signals (NLSs). In someaspects, at least one NLS is at or within 50 amino acids of theamino-terminus of the polynucleotide encoding a Cas9 or Cpflendonuclease and/or at least one NLS is at or within 50 amino acids ofthe carboxy-terminus of the polynucleotide encoding a Cas9 or Cpflendonuclease of the CRISPR/Cas system described herein. In some aspects,the polynucleotide of the CRISPR/Cas system described herein andencoding a Cas9 or Cpfl endonuclease is codon optimized for expressionin a eukaryotic cell.

Also provided herein is RNA encoding the single-molecule guide RNAdescribed herein.

Also provided herein is RNA encoding the CRISPR/Cas system describedherein.

Also provided herein is a DNA encoding the single-molecule guide RNAdescribed herein.

Also provided herein is a DNA encoding the CRISPR/Cas system describedherein.

Also provided herein is a vector comprising the DNA encoding thesingle-molecule guide RNA and CRISPR/Cas system. In some aspects, thevector is a plasmid. In some aspects, the vector is an AAV vectorparticle. In some aspects, the AAV vector particle is selected from thegroup consisting of any of those disclosed in SEQ ID NOs: 4734-5302 orTable 2.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of materials and methods disclosed and described in thisspecification can be better understood by reference to the accompanyingfigures, in which:

FIGS. 1A-B depict the type II CRISPR/Cas system;

FIG. 1A is a depiction of the type II CRISPR/Cas system including gRNA;

FIG. 1B is another depiction of the type II CRISPR/Cas system includingsgRNA;

FIGS. 2A-G describe the cutting efficiencies of S. pyogenes gRNAsselected via an in-vitro transcribed (IVT) gRNA screen in HEK293T cells;

FIG. 2A describes the cutting efficiencies in the range of 90.2 - 99.0%of S. pyogenes gRNAs selected via an in-vitro transcribed (IVT) gRNAscreen in HEK293T cells;

FIG. 2B describes the cutting efficiencies in the range of 84.0 - 89.9%of S. pyogenes gRNAs selected via an in-vitro transcribed (IVT) gRNAscreen in HEK293T cells;

FIG. 2C describes the cutting efficiencies in the range of 75.2 - 83.8%of S. pyogenes gRNAs selected via an in-vitro transcribed (IVT) gRNAscreen in HEK293T cells;

FIG. 2D describes the cutting efficiencies in the range of 63.9 - 74.6%of S. pyogenes gRNAs selected via an in-vitro transcribed (IVT) gRNAscreen in HEK293T cells;

FIG. 2E describes the cutting efficiencies in the range of 52.5 - 63.6%of S. pyogenes gRNAs selected via an in-vitro transcribed (IVT) gRNAscreen in HEK293T cells;

FIG. 2F describes the cutting efficiencies in the range of 33.6 - 52.1%of S. pyogenes gRNAs selected via an in-vitro transcribed (IVT) gRNAscreen in HEK293T cells;

FIG. 2G describes the cutting efficiencies in the range of 0 - 33.1% ofS. pyogenes gRNAs selected via an in-vitro transcribed (IVT) gRNA screenin HEK293T cells;

FIGS. 3A-C describe the cutting efficiency of S. pyogenes gRNAs inHEK293T cells;

FIG. 3A describes the cutting efficiency in the range of 82.3 - 99.0% ofS. pyogenes gRNAs in HEK293T cells;

FIG. 3B describes the cutting efficiency in the range of 58.2 - 81.4% ofS. pyogenes gRNAs in HEK293T cells; and

FIG. 3C describes the cutting efficiency in the range of 0 - 57.7% of S.pyogenes gRNAs in HEK293T cells.

FIGS. 4A-B describe the cutting efficiencies of S. aureus gRNAs selectedvia an in-vitro transcribed (IVT) gRNA screen in HEK293T cells;

FIG. 4A describes the cutting efficiencies in the range of 1.1 - 12.8%of S. aureus gRNAs selected via an in-vitro transcribed (IVT) gRNAscreen in HEK293T cells;

FIG. 4B describes the cutting efficiencies in the range of 0 - 1.0% ofS. aureus gRNAs selected via an in-vitro transcribed (IVT) gRNA screenin HEK293T cells; and

FIG. 5 describes the cutting efficiency in the range of 0 - 12.8% of S.aureus gRNAs in HEK293T cells.

BRIEF DESCRIPTION OF SEQUENCE LISTING

SEQ ID NOs: 1 - 620 are Cas endonuclease ortholog sequences.

SEQ ID NOs: 621 - 631 do not include sequences.

SEQ ID NOs: 632 - 4715 are microRNA sequences.

SEQ ID NOs: 4716 - 4733 do not include sequences.

SEQ ID NOs: 4734 - 5302 are AAV serotype sequences.

SEQ ID NO: 5303 is a DMPK nucleotide sequence.

SEQ ID NO: 5304 is a gene sequence including 1-5 kilobase pairs upstreamand/or downstream of the DMPK gene.

SEQ ID NOs: 5305 - 5332 are 20 bp spacer sequences for targeting withinor near a DMPK gene or other DNA sequence that encodes a regulatoryelement of the DMPK gene with a T. denticola Cas9 endonuclease.

SEQ ID NOs: 5333 - 5399 are 20 bp spacer sequences for targeting withinor near a DMPK gene or other DNA sequence that encodes a regulatoryelement of the DMPK gene with a S. thermophilus Cas9 endonuclease.

SEQ ID NOs: 5400 - 6048 are 20 bp spacer sequences for targeting withinor near a DMPK gene or other DNA sequence that encodes a regulatoryelement of the DMPK gene with a S. aureus Cas9 endonuclease.

SEQ ID NOs: 6049 - 6367 are 20 bp spacer sequences for targeting withinor near a DMPK gene or other DNA sequence that encodes a regulatoryelement of the DMPK gene with a N. meningitides Cas9 endonuclease.

SEQ ID NOs: 6368 - 15236 are 20 bp spacer sequences for targeting withinor near a DMPK gene or other DNA sequence that encodes a regulatoryelement of the DMPK gene with a S. pyogenes Cas9 endonuclease.

SEQ ID NOs: 15237 - 20697 are 20 bp spacer sequences for targetingwithin or near a DMPK gene or other DNA sequence that encodes aregulatory element of the DMPK gene with an Acidaminococcus, aLachnospiraceae, and a Franciscella Novicida Cpf1 endonuclease.

SEQ ID NOs: 20698 - 20727 do not include sequences.

SEQ ID NO: 20728 is a sample guide RNA (gRNA) for a S. pyogenes Cas9endonuclease.

SEQ ID NOs: 20729 - 20731 show sample sgRNA sequences.

DETAILED DESCRIPTION I. Introduction Genome Editing

The present disclosure provides strategies and techniques for thetargeted, specific alteration of the genetic information (genome) ofliving organisms. As used herein, the term “alteration” or “alterationof genetic information” refers to any change in the genome of a cell. Inthe context of treating genetic disorders, alterations may include, butare not limited to, insertion, deletion and correction. As used herein,the term “insertion” refers to an addition of one or more nucleotides ina DNA sequence. Insertions can range from small insertions of a fewnucleotides to insertions of large segments such as a cDNA or a gene.The term “deletion” refers to a loss or removal of one or morenucleotides in a DNA sequence or a loss or removal of the function of agene. In some cases, a deletion can include, for example, a loss of afew nucleotides, an exon, an intron, a gene segment, or the entiresequence of a gene. In some cases, deletion of a gene refers to theelimination or reduction of the function or expression of a gene or itsgene product. This can result from not only a deletion of sequenceswithin or near the gene, but also other events (e.g., insertion,nonsense mutation) that disrupt the expression of the gene. The term“correction” as used herein, refers to a change of one or morenucleotides of a genome in a cell, whether by insertion, deletion orsubstitution. Such correction may result in a more favorable genotypicor phenotypic outcome, whether in structure or function, to the genomicsite which was corrected. One non-limiting example of a “correction”includes the correction of a mutant or defective sequence to a wild-typesequence which restores structure or function to a gene or its geneproduct(s). Depending on the nature of the mutation, correction may beachieved via various strategies disclosed herein. In one non-limitingexample, a missense mutation may be corrected by replacing the regioncontaining the mutation with its wild-type counterpart. As anotherexample, duplication mutations (e.g., repeat expansions) in a gene maybe corrected by removing the extra sequences.

In some aspects, alterations may also include a gene knock-in, knock-outor knock-down. As used herein, the term “knock-in” refers to an additionof a DNA sequence, or fragment thereof into a genome. Such DNA sequencesto be knocked-in may include an entire gene or genes, may includeregulatory sequences associated with a gene or any portion or fragmentof the foregoing. For example, a cDNA encoding the wild-type protein maybe inserted into the genome of a cell carrying a mutant gene. Knock-instrategies need not replace the defective gene, in whole or in part. Insome cases, a knock-in strategy may further involve substitution of anexisting sequence with the provided sequence, e.g., substitution of amutant allele with a wild-type copy. On the other hand, the term“knock-out” refers to the elimination of a gene or the expression of agene. For example, a gene can be knocked out by either a deletion or anaddition of a nucleotide sequence that leads to a disruption of thereading frame. As another example, a gene may be knocked out byreplacing a part of the gene with an irrelevant sequence. Finally, theterm “knock-down” as used herein refers to reduction in the expressionof a gene or its gene product(s). As a result of a gene knock-down, theprotein activity or function may be attenuated or the protein levels maybe reduced or eliminated.

Genome editing generally refers to the process of modifying thenucleotide sequence of a genome, preferably in a precise orpre-determined manner. Examples of methods of genome editing describedherein include methods of using site-directed nucleases to cutdeoxyribonucleic acid (DNA) at precise target locations in the genome,thereby creating single-strand or double-strand DNA breaks at particularlocations within the genome. Such breaks can be and regularly arerepaired by natural, endogenous cellular processes, such ashomology-directed repair (HDR) and non-homologous end joining (NHEJ), asreviewed in Cox et al., Nature Medicine 21(2), 121-31 (2015). These twomain DNA repair processes consist of a family of alternative pathways.NHEJ directly joins the DNA ends resulting from a double-strand break,sometimes with the loss or addition of nucleotide sequence, which maydisrupt or enhance gene expression. HDR utilizes a homologous sequence,or donor sequence, as a template for inserting a defined DNA sequence atthe break point. The homologous sequence can be in the endogenousgenome, such as a sister chromatid. Alternatively, the donor can be anexogenous nucleic acid, such as a plasmid, a single-strandoligonucleotide, a double-stranded oligonucleotide, a duplexoligonucleotide or a virus, that has regions of high homology with thenuclease-cleaved locus, but which can also contain additional sequenceor sequence changes including deletions that can be incorporated intothe cleaved target locus. A third repair mechanism can bemicrohomology-mediated end joining (MMEJ), also referred to as“Alternative NHEJ,” in which the genetic outcome is similar to NHEJ inthat small deletions and insertions can occur at the cleavage site. MMEJcan make use of homologous sequences of a few base pairs flanking theDNA break site to drive a more favored DNA end joining repair outcome,and recent reports have further elucidated the molecular mechanism ofthis process; see, e.g., Cho and Greenberg, Nature 518, 174-76 (2015);Kent et al., Nature Structural and Molecular Biology, Adv. Onlinedoi:10.1038/nsmb.2961(2015); Mateos-Gomez et al., Nature 518, 254-57(2015); Ceccaldi et al., Nature 528, 258-62 (2015). In some instances,it may be possible to predict likely repair outcomes based on analysisof potential microhomologies at the site of the DNA break.

Each of these genome editing mechanisms can be used to create desiredgenomic alterations. A step in the genome editing process can be tocreate one or two DNA breaks, the latter as double-strand breaks or astwo single-stranded breaks, in the target locus as near the site ofintended mutation. This can be achieved via the use of site-directedpolypeptides, as described and illustrated herein.

CRISPR Endonuclease System

A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)genomic locus can be found in the genomes of many prokaryotes (e.g.,bacteria and archaea). In prokaryotes, the CRISPR locus encodes productsthat function as a type of immune system to help defend the prokaryotesagainst foreign invaders, such as virus and phage. There are threestages of CRISPR locus function: integration of new sequences into theCRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreigninvader nucleic acid. Five types of CRISPR systems (e.g., Type I, TypeII, Type III, Type U, and Type V) have been identified.

A CRISPR locus includes a number of short repeating sequences referredto as “repeats.” When expressed, the repeats can form secondarystructures (e.g., hairpins) and/or comprise unstructured single-strandedsequences. The repeats usually occur in clusters and frequently divergebetween species. The repeats are regularly interspaced with uniqueintervening sequences referred to as “spacers,” resulting in arepeat-spacer-repeat locus architecture. The spacers are identical to orhave high homology with known foreign invader sequences. A spacer-repeatunit encodes a crisprRNA (crRNA), which is processed into a mature formof the spacer-repeat unit. A crRNA comprises a “seed” or spacer sequencethat is involved in targeting a target nucleic acid (in the naturallyoccurring form in prokaryotes, the spacer sequence targets the foreigninvader nucleic acid). A spacer sequence is located at the 5′ or 3′ endof the crRNA.

A CRISPR locus also comprises polynucleotide sequences encoding CRISPRAssociated (Cas) genes. Cas genes encode endonucleases involved in thebiogenesis and the interference stages of crRNA function in prokaryotes.Some Cas genes comprise homologous secondary and/or tertiary structures.

Type II CRISPR Systems

crRNA biogenesis in a Type II CRISPR system in nature requires atrans-activating CRISPR RNA (tracrRNA). Non-limiting examples of Type IICRISPR systems are shown in FIGS. 1A and 1B. The tracrRNA can bemodified by endogenous RNaseIII, and then hybridizes to a crRNA repeatin the pre-crRNA array. Endogenous RNaseIII can be recruited to cleavethe pre-crRNA. Cleaved crRNAs can be subjected to exoribonucleasetrimming to produce the mature crRNA form (e.g., 5′ trimming). ThetracrRNA can remain hybridized to the crRNA, and the tracrRNA and thecrRNA associate with a site-directed polypeptide (e.g., Cas9). The crRNAof the crRNA-tracrRNA-Cas9 complex can guide the complex to a targetnucleic acid to which the crRNA can hybridize. Hybridization of thecrRNA to the target nucleic acid can activate Cas9 for targeted nucleicacid cleavage. The target nucleic acid in a Type II CRISPR system isreferred to as a protospacer adjacent motif (PAM). In nature, the PAM isessential to facilitate binding of a site-directed polypeptide (e.g.,Cas9) to the target nucleic acid. Type II systems (also referred to asNmeni or CASS4) are further subdivided into Type II-A (CASS4) and II-B(CASS4a). Jinek et al., Science, 337(6096):816-821 (2012) showed thatthe CRISPR/Cas9 system is useful for RNA-programmable genome editing,and international patent application publication number WO2013/176772provides numerous examples and applications of the CRISPR/Casendonuclease system for site-specific gene editing.

Type V CRISPR Systems

Type V CRISPR systems have several important differences from Type IIsystems. For example, Cpfl is a single RNA-guided endonuclease that, incontrast to Type II systems, lacks tracrRNA. In fact, Cpfl-associatedCRISPR arrays can be processed into mature crRNAs without therequirement of an additional trans-activating tracrRNA. The Type VCRISPR array can be processed into short mature crRNAs of 42-44nucleotides in length, with each mature crRNA beginning with 19nucleotides of direct repeat followed by 23-25 nucleotides of spacersequence. In contrast, mature crRNAs in Type II systems can start with20-24 nucleotides of spacer sequence followed by about 22 nucleotides ofdirect repeat. Also, Cpfl can utilize a T-rich protospacer-adjacentmotif such that Cpfl-crRNA complexes efficiently cleave target DNApreceded by a short T-rich PAM, which is in contrast to the G-rich PAMfollowing the target DNA for Type II systems. Thus, Type V systemscleave at a point that is distant from the PAM, while Type II systemscleave at a point that is adjacent to the PAM. In addition, in contrastto Type II systems, Cpfl cleaves DNA via a staggered DNA double-strandedbreak with a 4 or 5 nucleotide 5′ overhang. Type II systems cleave via ablunt double-stranded break. Similar to Type II systems, Cpfl contains apredicted RuvC-like endonuclease domain, but lacks a second HNHendonuclease domain, which is in contrast to Type II systems.

Cas Genes/Polypeptides and Protospacer Adjacent Motifs

Exemplary CRISPR/Cas polypeptides include the Cas9 polypeptides aspublished in Fonfara et al., Nucleic Acids Research, 42: 2577-2590(2014). The CRISPR/Cas gene naming system has undergone extensiverewriting since the Cas genes were discovered. Fonfara et al. alsoprovides PAM sequences for the Cas9 polypeptides from various species(see also Table 1 infra).

II. Compositions and Methods of the Disclosure

Provided herein are cellular, ex vivo and in vivo methods for usinggenome engineering tools to create permanent changes to the genomeby: 1) deleting the abnormal repeat expansion within or near the DMPKgene, by inducing two double-stranded DNA breaks at both sides of theexpanded region; 2) deleting the abnormal repeat expansion (in whole orin part) within or near the DMPK gene, by inducing one double-strandedDNA break proximal to the expanded region; 3) deleting or mutating theDMPK gene by inducing one or more insertions or deletions within or nearthe DMPK gene or other DNA sequences that encode regulatory elements ofthe DMPK gene; 4) deleting the mutant DMPK gene and inserting awild-type DMPK gene, a cDNA or a minigene (comprised of one or moreexons and introns or natural or synthetic introns) into the DMPK genelocus or a safe harbor locus; or 5) targeting a dCas9 fused to chromatinmodifying proteins to DMPK locus to prevent production of transcriptswith expanded repeats. Such methods use endonucleases, such asCRISPR-associated (Cas9, Cpfl and the like) nucleases, to permanentlyedit one or more mutations within or near the genomic locus of the DMPKgene or other DNA sequences that encode regulatory elements of the DMPKgene. In this way, examples set forth in the present disclosure can helpto restore the wild-type sequence or similar DMPK non-coding sequenceof, or otherwise reduce or eliminate the expression of, the DMPK gene orthe aberrant transcripts with expanded repeats with as few as a singletreatment (rather than deliver potential therapies for the lifetime ofthe patient).

Site-Directed Polypeptides (Endonucleases, Enzymes)

A site-directed polypeptide is a nuclease used in genome editing tocleave DNA. The site-directed polypeptide can be administered to a cellor a patient as either: one or more polypeptides, or one or more mRNAsencoding the polypeptide. Any of the enzymes or orthologs listed in SEQID NOs: 1-620, or disclosed herein, may be utilized in the methodsherein.

In the context of a CRISPR/Cas9 or CRISPR/Cpfl system, the site-directedpolypeptide can bind to a guide RNA that, in turn, specifies the site inthe target DNA to which the polypeptide is directed. In the CRISPR/Cas9or CRISPR/Cpfl systems disclosed herein, the site-directed polypeptidecan be an endonuclease, such as a DNA endonuclease.

A site-directed polypeptide can comprise a plurality of nucleicacid-cleaving (i.e., nuclease) domains. Two or more nucleicacid-cleaving domains can be linked together via a linker. For example,the linker can comprise a flexible linker. Linkers can comprise 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40 or more amino acids in length.

Naturally-occurring wild-type Cas9 enzymes comprise two nucleasedomains, a HNH nuclease domain and a RuvC domain. Herein, the term“Cas9” refers to both a naturally-occurring and a recombinant Cas9. Cas9enzymes contemplated herein can comprise a HNH or HNH-like nucleasedomain, and/or a RuvC or RuvC-like nuclease domain.

HNH or HNH-like domains comprise a McrA-like fold. HNH or HNH-likedomains comprises two antiparallel β-strands and an α-helix. HNH orHNH-like domains comprises a metal binding site (e.g., a divalent cationbinding site). HNH or HNH-like domains can cleave one strand of a targetnucleic acid (e.g., the complementary strand of the crRNA targetedstrand).

RuvC or RuvC-like domains comprise an RNaseH or RNaseH-like fold.RuvC/RNaseH domains are involved in a diverse set of nucleic acid-basedfunctions including acting on both RNA and DNA. The RNaseH domaincomprises 5 β-strands surrounded by a plurality of α-helices.RuvC/RNaseH or RuvC/RNaseH-like domains comprise a metal binding site(e.g., a divalent cation binding site). RuvC/RNaseH or RuvC/RNaseH-likedomains can cleave one strand of a target nucleic acid (e.g., thenon-complementary strand of a double-stranded target DNA).

Site-directed polypeptides can introduce double-strand breaks orsingle-strand breaks in nucleic acids, e.g., genomic DNA. Thedouble-strand break can stimulate a cell’s endogenous DNA-repairpathways (e.g., homology-dependent repair (HDR) or NHEJ or alternativenon-homologous end joining (A-NHEJ) or microhomology-mediated endjoining (MMEJ)). NHEJ can repair cleaved target nucleic acid without theneed for a homologous template. This can sometimes result in smalldeletions or insertions (indels) in the target nucleic acid at the siteof cleavage, and can lead to disruption or alteration of geneexpression. HDR can occur when a homologous repair template, or donor,is available. The homologous donor template can comprise sequences thatare homologous to sequences flanking the target nucleic acid cleavagesite. The sister chromatid can be used by the cell as the repairtemplate. However, for the purposes of genome editing, the repairtemplate can be supplied as an exogenous nucleic acid, such as aplasmid, duplex oligonucleotide, single-strand oligonucleotide or viralnucleic acid. With exogenous donor templates, an additional nucleic acidsequence (such as a transgene) or modification (such as a single ormultiple base change or a deletion) can be introduced between theflanking regions of homology so that the additional or altered nucleicacid sequence also becomes incorporated into the target locus. MMEJ canresult in a genetic outcome that is similar to NHEJ in that smalldeletions and insertions can occur at the cleavage site. MMEJ can makeuse of homologous sequences of a few base pairs flanking the cleavagesite to drive a favored end-joining DNA repair outcome. In someinstances, it may be possible to predict likely repair outcomes based onanalysis of potential microhomologies in the nuclease target regions.

Thus, in some cases, homologous recombination can be used to insert anexogenous polynucleotide sequence into the target nucleic acid cleavagesite. An exogenous polynucleotide sequence is termed a “donorpolynucleotide” (or donor or donor sequence) herein. The donorpolynucleotide, a portion of the donor polynucleotide, a copy of thedonor polynucleotide, or a portion of a copy of the donor polynucleotidecan be inserted into the target nucleic acid cleavage site. The donorpolynucleotide can be an exogenous polynucleotide sequence, i.e., asequence that does not naturally occur at the target nucleic acidcleavage site.

The modifications of the target DNA due to NHEJ and/or HDR can lead to,for example, mutations, deletions, alterations, integrations, genecorrection, gene replacement, gene tagging, transgene insertion,nucleotide deletion, gene disruption, translocations and/or genemutation. The processes of deleting genomic DNA and integratingnon-native nucleic acid into genomic DNA are examples of genome editing.

The site-directed polypeptide can comprise an amino acid sequence havingat least 10%, at least 15%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%, or 100% amino acidsequence identity to a wild-type exemplary site-directed polypeptide[e.g., Cas9 from S. pyogenes, US2014/0068797 Sequence ID No. 8 orSapranauskas et al., Nucleic Acids Res, 39(21): 9275-9282 (2011)], andvarious other site-directed polypeptides. The site-directed polypeptidecan comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identityto a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes,supra) over 10 contiguous amino acids. The site-directed polypeptide cancomprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to awild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra)over 10 contiguous amino acids. The site-directed polypeptide cancomprise at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to awild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra)over 10 contiguous amino acids in a HNH nuclease domain of thesite-directed polypeptide. The site-directed polypeptide can comprise atmost: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-typesite-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10contiguous amino acids in a HNH nuclease domain of the site-directedpolypeptide. The site-directed polypeptide can comprise at least: 70,75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-typesite-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10contiguous amino acids in a RuvC nuclease domain of the site-directedpolypeptide. The site-directed polypeptide can comprise at most: 70, 75,80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directedpolypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguousamino acids in a RuvC nuclease domain of the site-directed polypeptide.

The site-directed polypeptide can comprise a modified form of awild-type exemplary site-directed polypeptide. The modified form of thewild- type exemplary site-directed polypeptide can comprise a mutationthat reduces the nucleic acid-cleaving activity of the site-directedpolypeptide. The modified form of the wild-type exemplary site-directedpolypeptide can have less than 90%, less than 80%, less than 70%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20%,less than 10%, less than 5%, or less than 1% of the nucleicacid-cleaving activity of the wild-type exemplary site-directedpolypeptide (e.g., Cas9 from S. pyogenes, supra). The modified form ofthe site-directed polypeptide can have no substantial nucleicacid-cleaving activity. When a site-directed polypeptide is a modifiedform that has no substantial nucleic acid-cleaving activity, it isreferred to herein as “enzymatically inactive.”

The modified form of the site-directed polypeptide can comprise amutation such that it can induce a single-strand break (SSB) on a targetnucleic acid (e.g., by cutting only one of the sugar-phosphate backbonesof a double-strand target nucleic acid). In some aspects, the mutationcan result in less than 90%, less than 80%, less than 70%, less than60%, less than 50%, less than 40%, less than 30%, less than 20%, lessthan 10%, less than 5%, or less than 1% of the nucleic acid-cleavingactivity in one or more of the plurality of nucleic acid-cleavingdomains of the wild-type site directed polypeptide (e.g., Cas9 from S.pyogenes, supra). In some aspects, the mutation can result in one ormore of the plurality of nucleic acid-cleaving domains retaining theability to cleave the complementary strand of the target nucleic acid,but reducing its ability to cleave the non-complementary strand of thetarget nucleic acid. The mutation can result in one or more of theplurality of nucleic acid-cleaving domains retaining the ability tocleave the non-complementary strand of the target nucleic acid, butreducing its ability to cleave the complementary strand of the targetnucleic acid. For example, residues in the wild-type exemplary S.pyogenes Cas9 polypeptide, such as Asp10, His840, Asn854 and Asn856, aremutated to inactivate one or more of the plurality of nucleicacid-cleaving domains (e.g., nuclease domains). The residues to bemutated can correspond to residues Asp10, His840, Asn854 and Asn856 inthe wild-type exemplary S. pyogenes Cas9 polypeptide (e.g., asdetermined by sequence and/or structural alignment). Non-limitingexamples of mutations include D10A, H840A, N854A or N856A. One skilledin the art will recognize that mutations other than alaninesubstitutions can be suitable.

In some aspects, a D10A mutation can be combined with one or more ofH840A, N854A, or N856A mutations to produce a site-directed polypeptidesubstantially lacking DNA cleavage activity. A H840A mutation can becombined with one or more of D10A, N854A, or N856A mutations to producea site-directed polypeptide substantially lacking DNA cleavage activity.A N854A mutation can be combined with one or more of H840A, D10A, orN856A mutations to produce a site-directed polypeptide substantiallylacking DNA cleavage activity. A N856A mutation can be combined with oneor more of H840A, N854A, or D10A mutations to produce a site-directedpolypeptide substantially lacking DNA cleavage activity. Site-directedpolypeptides that comprise one substantially inactive nuclease domainare referred to as “nickases.”

Nickase variants of RNA-guided endonucleases, for example Cas9, can beused to increase the specificity of CRISPR-mediated genome editing. Wildtype Cas9 is typically guided by a single guide RNA designed tohybridize with a specified ~20 nucleotide sequence in the targetsequence (such as an endogenous genomic locus). However, severalmismatches can be tolerated between the guide RNA and the target locus,effectively reducing the length of required homology in the target siteto, for example, as little as 13 nt of homology, and thereby resultingin elevated potential for binding and double-strand nucleic acidcleavage by the CRISPR/Cas9 complex elsewhere in the target genome -also known as off-target cleavage. Because nickase variants of Cas9 eachonly cut one strand, in order to create a double-strand break it isnecessary for a pair of nickases to bind in close proximity and onopposite strands of the target nucleic acid, thereby creating a pair ofnicks, which is the equivalent of a double-strand break. This requiresthat two separate guide RNAs – one for each nickase – must bind in closeproximity and on opposite strands of the target nucleic acid. Thisrequirement essentially doubles the minimum length of homology neededfor the double-strand break to occur, thereby reducing the likelihoodthat a double-strand cleavage event will occur elsewhere in the genome,where the two guide RNA sites – if they exist – are unlikely to besufficiently close to each other to enable the double-strand break toform. As described in the art, nickases can also be used to promote HDRversus NHEJ. HDR can be used to introduce selected changes into targetsites in the genome through the use of specific donor sequences thateffectively mediate the desired changes.

Mutations contemplated can include substitutions, additions, anddeletions, or any combination thereof. The mutation converts the mutatedamino acid to alanine. The mutation converts the mutated amino acid toanother amino acid (e.g., glycine, serine, threonine, cysteine, valine,leucine, isoleucine, methionine, proline, phenylalanine, tyrosine,tryptophan, aspartic acid, glutamic acid, asparagine, glutamine,histidine, lysine, or arginine). The mutation converts the mutated aminoacid to a non-natural amino acid (e.g., selenomethionine). The mutationconverts the mutated amino acid to amino acid mimics (e.g.,phosphomimics). The mutation can be a conservative mutation. Forexample, the mutation converts the mutated amino acid to amino acidsthat resemble the size, shape, charge, polarity, conformation, and/orrotamers of the mutated amino acids (e.g., cysteine/serine mutation,lysine/asparagine mutation, histidine/phenylalanine mutation). Themutation can cause a shift in reading frame and/or the creation of apremature stop codon. Mutations can cause changes to regulatory regionsof genes or loci that affect expression of one or more genes.

The site-directed polypeptide (e.g., variant, mutated, enzymaticallyinactive and/or conditionally enzymatically inactive site-directedpolypeptide) can target nucleic acid. The site-directed polypeptide(e.g., variant, mutated, enzymatically inactive and/or conditionallyenzymatically inactive endoribonuclease) can target DNA. Thesite-directed polypeptide (e.g., variant, mutated, enzymaticallyinactive and/or conditionally enzymatically inactive endoribonuclease)can target RNA.

The site-directed polypeptide can comprise one or more non-nativesequences (e.g., the site-directed polypeptide is a fusion protein).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), a nucleic acid binding domain, and two nucleic acidcleaving domains (i.e., a HNH domain and a RuvC domain).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains, wherein oneor both of the nucleic acid cleaving domains comprise at least 50% aminoacid identity to a nuclease domain from Cas9 from a bacterium (e.g., S.pyogenes).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), and a non-native sequence (for example, anuclear localization signal) or a linker linking the site-directedpolypeptide to a non-native sequence.

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), wherein the site-directed polypeptidecomprises a mutation in one or both of the nucleic acid cleaving domainsthat reduces the cleaving activity of the nuclease domains by at least50%.

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), wherein one of the nuclease domains comprisesmutation of aspartic acid 10, and/or wherein one of the nuclease domainscan comprise a mutation of histidine 840, and wherein the mutationreduces the cleaving activity of the nuclease domain(s) by at least 50%.

The one or more site-directed polypeptides, e.g. DNA endonucleases, cancomprise two nickases that together effect one double-strand break at aspecific locus in the genome, or four nickases that together effect orcause two double-strand breaks at specific loci in the genome.Alternatively, one site-directed polypeptide, e.g. DNA endonuclease, caneffect or cause one double-strand break at a specific locus in thegenome.

Non-limiting examples of Cas9 orthologs from other bacterial strainsinclude but are not limited to, Cas proteins identified in Acaryochlorismarina MBIC11017; Acetohalobium arabaticum DSM 5501; Acidithiobacilluscaldus; Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillusacidocaldarius LAA1; Alicyclobacillus acidocaldarius subsp.acidocaldarius DSM 446; Allochromatium vinosum DSM 180; Ammoniƒexdegensii KC4; Anabaena variabilis ATCC 29413; Arthrospira maxima CS-328;Arthrospira platensis str. Paraca; Arthrospira sp. PCC 8005; Bacilluspseudomycoides DSM 12442; Bacillus selenitireducens MLS10;Burkholderiales bacterium 1_1_47; Caldicelulosiruptor becscii DSM 6725;Candidatus Desulƒorudis audaxviator MP104C; Caldicellulosiruptorhydrothermalis _108; Clostridium phage c-st; Clostridium botulinum A3str. Loch Maree; Clostridium botulinum Ba4 str. 657; Clostridiumdifficile QCD-63q42; Crocosphaera watsonii WH 8501; Cyanothece sp. ATCC51142; Cyanothece sp. CCY0110; Cyanothece sp. PCC 7424; Cyanothece sp.PCC 7822; Exiguobacterium sibiricum 255-15; Finegoldia magna ATCC 29328;Ktedonobacter racemifer DSM 44963; Lactobacillus delbrueckii subsp.bulgaricus PB2003/044-T3-4; Lactobacillus salivarius ATCC 11741;Listeria innocua; Lyngbya sp. PCC 8106; Marinobacter sp. ELB17;Methanohalobium evestigatum Z-7303; Microcystis phage Ma-LMM01;Microcystis aeruginosa NIES-843; Microscilla marina ATCC 23134;Microcoleus chthonoplastes PCC 7420; Neisseria meningitidis;Nitrosococcus halophilus Nc4; Nocardiopsis dassonvillei subsp.dassonvillei DSM 43111; Nodularia spumigena CCY9414; Nostoc sp. PCC7120; Oscillatoria sp. PCC 6506; Pelotomaculum_ thermopropionicum_SI;Petrotoga mobilis SJ95; Polaromonas naphthalenivorans CJ2; Polaromonassp. JS666; Pseudoalteromonas haloplanktis TAC125; Streptomycespristinaespiralis ATCC 25486; Streptomyces pristinaespiralis ATCC 25486;Streptococcus thermophilus; Streptomyces viridochromogenes DSM 40736;Streptosporangium roseum DSM 43021; Synechococcus sp. PCC 7335; andThermosipho africanus TCF52B (Chylinski et al., RNA Biol., 2013; 10(5):726-737).

In addition to Cas9 orthologs, other Cas9 variants such as fusionproteins of inactive dCas9 and effector domains with different functionsmay be served as a platform for genetic modulation. Any of the foregoingenzymes may be useful in the present disclosure.

Further examples of endonucleases which may be utilized in the presentdisclosure are given in SEQ ID NOs: 1-620. These proteins may bemodified before use or may be encoded in a nucleic acid sequence such asa DNA, RNA or mRNA or within a vector construct such as the plasmids orAAV vectors taught herein. Further, they may be codon optimized.

SEQ ID NOs: 1-620 disclose a non-exhaustive listing of endonucleasesequences.

Genome-Targeting Nucleic Acid

The present disclosure provides a genome-targeting nucleic acid that candirect the activities of an associated polypeptide (e.g., asite-directed polypeptide) to a specific target sequence within a targetnucleic acid. The genome-targeting nucleic acid can be an RNA. Agenome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein. Aguide RNA can comprise at least a spacer sequence that hybridizes to atarget nucleic acid sequence of interest, and a CRISPR repeat sequence.In Type II systems, the gRNA also comprises a second RNA called thetracrRNA sequence. In the Type II guide RNA (gRNA), the CRISPR repeatsequence and tracrRNA sequence hybridize to each other to form a duplex.In the Type V guide RNA (gRNA), the crRNA forms a duplex. In bothsystems, the duplex can bind a site-directed polypeptide, such that theguide RNA and site-direct polypeptide form a complex. Thegenome-targeting nucleic acid can provide target specificity to thecomplex by virtue of its association with the site-directed polypeptide.The genome-targeting nucleic acid thus can direct the activity of thesite-directed polypeptide.

Exemplary guide RNAs include the spacer sequences in SEQ ID NOs:5305-20697 of the Sequence Listing. As is understood by the person ofordinary skill in the art, each guide RNA can be designed to include aspacer sequence complementary to its genomic target sequence. Forexample, each of the spacer sequences in SEQ ID NOs: 5305-20697 of theSequence Listing can be put into a single RNA chimera or a crRNA (alongwith a corresponding tracrRNA). See Jinek et al., Science, 337, 816-821(2012) and Deltcheva et al., Nature, 471, 602-607 (2011).

The genome-targeting nucleic acid can be a double-molecule guide RNA.The genome-targeting nucleic acid can be a single-molecule guide RNA.

A double-molecule guide RNA can comprise two strands of RNA. The firststrand comprises in the 5’ to 3’ direction, an optional spacer extensionsequence, a spacer sequence and a minimum CRISPR repeat sequence. Thesecond strand can comprise a minimum tracrRNA sequence (complementary tothe minimum CRISPR repeat sequence), a 3’ tracrRNA sequence and anoptional tracrRNA extension sequence.

A single-molecule guide RNA (sgRNA) in a Type II system can comprise, inthe 5’ to 3’ direction, an optional spacer extension sequence, a spacersequence, a minimum CRISPR repeat sequence, a single-molecule guidelinker, a minimum tracrRNA sequence, a 3’ tracrRNA sequence and anoptional tracrRNA extension sequence. The optional tracrRNA extensioncan comprise elements that contribute additional functionality (e.g.,stability) to the guide RNA. The single-molecule guide linker can linkthe minimum CRISPR repeat and the minimum tracrRNA sequence to form ahairpin structure. The optional tracrRNA extension can comprise one ormore hairpins.

The sgRNA can comprise a 20 nucleotide spacer sequence at the 5’ end ofthe sgRNA sequence. The sgRNA can comprise a less than a 20 nucleotidespacer sequence at the 5’ end of the sgRNA sequence. The sgRNA cancomprise a more than 20 nucleotide spacer sequence at the 5’ end of thesgRNA sequence. The sgRNA can comprise a variable length spacer sequencewith 17-30 nucleotides at the 5’ end of the sgRNA sequence (see Table1).

The sgRNA can comprise no uracil at the 3’end of the sgRNA sequence,such as in SEQ ID NO: 20730 of Table 1. The sgRNA can comprise one ormore uracil at the 3’end of the sgRNA sequence, such as in SEQ ID NO:20731 in Table 1. For example, the sgRNA can comprise 1 uracil (U) atthe 3’ end of the sgRNA sequence. The sgRNA can comprise 2 uracil (UU)at the 3’ end of the sgRNA sequence. The sgRNA can comprise 3 uracil(UUU) at the 3’ end of the sgRNA sequence. The sgRNA can comprise 4uracil (UUUU) at the 3’ end of the sgRNA sequence. The sgRNA cancomprise 5 uracil (UUUUU) at the 3’ end of the sgRNA sequence. The sgRNAcan comprise 6 uracil (UUUUUU) at the 3‘ end of the sgRNA sequence. ThesgRNA can comprise 7 uracil (UUUUUUU) at the 3‘ end of the sgRNAsequence. The sgRNA can comprise 8 uracil (UUUUUUUU) at the 3‘ end ofthe sgRNA sequence.

The sgRNA can be unmodified or modified. For example, modified sgRNAscan comprise one or more 2’-O-methyl phosphorothioate nucleotides.

TABLE 1 SEQ ID NO. sgRNA sequence 20729nnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu 20730nnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugc 20731n₍₁₇₋₃₀₎guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcu₍₁₋₈₎

A single-molecule guide RNA (sgRNA) in a Type V system can comprise, inthe 5’ to 3’ direction, a minimum CRISPR repeat sequence and a spacersequence.

By way of illustration, guide RNAs used in the CRISPR/Cas9 orCRISPR/Cpfl system, or other smaller RNAs can be readily synthesized bychemical means, as illustrated below and described in the art. Whilechemical synthetic procedures are continually expanding, purificationsof such RNAs by procedures such as high performance liquidchromatography (HPLC, which avoids the use of gels such as PAGE) tendsto become more challenging as polynucleotide lengths increasesignificantly beyond a hundred or so nucleotides. One approach used forgenerating RNAs of greater length is to produce two or more moleculesthat are ligated together. Much longer RNAs, such as those encoding aCas9 or Cpfl endonuclease, are more readily generated enzymatically.Various types of RNA modifications can be introduced during or afterchemical synthesis and/or enzymatic generation of RNAs, e.g.,modifications that enhance stability, reduce the likelihood or degree ofinnate immune response, and/or enhance other attributes, as described inthe art.

Spacer Extension Sequence

In some examples of genome-targeting nucleic acids, a spacer extensionsequence can modify activity, provide stability and/or provide alocation for modifications of a genome-targeting nucleic acid. A spacerextension sequence can modify on- or off-target activity or specificity.In some examples, a spacer extension sequence can be provided. Thespacer extension sequence can have a length of more than 1, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000,4000, 5000, 6000, or 7000 or more nucleotides. The spacer extensionsequence can have a length of less than 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260,280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000,7000 or more nucleotides. The spacer extension sequence can be less than10 nucleotides in length. The spacer extension sequence can be between10-30 nucleotides in length. The spacer extension sequence can bebetween 30-70 nucleotides in length.

The spacer extension sequence can comprise another moiety (e.g., astability control sequence, an endoribonuclease binding sequence, aribozyme). The moiety can decrease or increase the stability of anucleic acid targeting nucleic acid. The moiety can be a transcriptionalterminator segment (i.e., a transcription termination sequence). Themoiety can function in a eukaryotic cell. The moiety can function in aprokaryotic cell. The moiety can function in both eukaryotic andprokaryotic cells. Non-limiting examples of suitable moieties include: a5’ cap (e.g., a 7-methylguanylate cap (m7 G)), a riboswitch sequence(e.g., to allow for regulated stability and/or regulated accessibilityby proteins and protein complexes), a sequence that forms a dsRNA duplex(i.e., a hairpin), a sequence that targets the RNA to a subcellularlocation (e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like).

Spacer Sequence

The spacer sequence hybridizes to a sequence in a target nucleic acid ofinterest. The spacer of a genome-targeting nucleic acid can interactwith a target nucleic acid in a sequence-specific manner viahybridization (i.e., base pairing). The nucleotide sequence of thespacer can vary depending on the sequence of the target nucleic acid ofinterest.

In a CRISPR/Cas system herein, the spacer sequence can be designed tohybridize to a target nucleic acid that is located 5’ of a PAM of theCas9 enzyme used in the system. The spacer may perfectly match thetarget sequence or may have mismatches. Each Cas9 enzyme has aparticular PAM sequence that it recognizes in a target DNA. For example,S. pyogenes recognizes in a target nucleic acid a PAM that comprises thesequence 5’-NRG-3’, where R comprises either A or G, where N is anynucleotide and N is immediately 3’ of the target nucleic acid sequencetargeted by the spacer sequence.

The target nucleic acid sequence can comprise 20 nucleotides. The targetnucleic acid can comprise less than 20 nucleotides. The target nucleicacid can comprise more than 20 nucleotides. The target nucleic acid cancomprise at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30or more nucleotides. The target nucleic acid can comprise at most: 5,10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.The target nucleic acid sequence can comprise 20 bases immediately 5’ ofthe first nucleotide of the PAM. For example, in a sequence comprising5’-NNNNNNNNNNNNNNNNNNNNRG-3’ (SEQ ID NO: 20728), the target nucleic acidcan comprise the sequence that corresponds to the Ns, wherein N is anynucleotide, and the underlined NRG sequence is the S. pyogenes PAM. Thistarget nucleic acid sequence is often referred to as the PAM strand, andthe complementary nucleic acid sequence is often referred to the non-PAMstrand. One of skill in the art would recognize that the spacer sequencehybridizes to the non-PAM strand of the target nucleic acid (FIGS. 1Aand 1B).

The spacer sequence that hybridizes to the target nucleic acid can havea length of at least about 6 nucleotides (nt). The spacer sequence canbe at least about 6 nt, at least about 10 nt, at least about 15 nt, atleast about 18 nt, at least about 19 nt, at least about 20 nt, at leastabout 25 nt, at least about 30 nt, at least about 35 nt or at leastabout 40 nt, from about 6 nt to about 80 nt, from about 6 nt to about 50nt, from about 6 nt to about 45 nt, from about 6 nt to about 40 nt, fromabout 6 nt to about 35 nt, from about 6 nt to about 30 nt, from about 6nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt toabout 19 nt, from about 10 nt to about 50 nt, from about 10 nt to about45 nt, from about 10 nt to about 40 nt, from about 10 nt to about 35 nt,from about 10 nt to about 30 nt, from about 10 nt to about 25 nt, fromabout 10 nt to about 20 nt, from about 10 nt to about 19 nt, from about19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 ntto about 35 nt, from about 19 nt to about 40 nt, from about 19 nt toabout 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt,from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, fromabout 20 nt to about 45 nt, from about 20 nt to about 50 nt, or fromabout 20 nt to about 60 nt. In some examples, the spacer sequence cancomprise 20 nucleotides. In some examples, the spacer can comprise 19nucleotides. In some examples, the spacer can comprise 18 nucleotides.In some examples, the spacer can comprise 22 nucleotides.

In some examples, the percent complementarity between the spacersequence and the target nucleic acid is at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, at least about 99%, or 100%. In some examples, thepercent complementarity between the spacer sequence and the targetnucleic acid is at most about 30%, at most about 40%, at most about 50%,at most about 60%, at most about 65%, at most about 70%, at most about75%, at most about 80%, at most about 85%, at most about 90%, at mostabout 95%, at most about 97%, at most about 98%, at most about 99%, or100%. In some examples, the percent complementarity between the spacersequence and the target nucleic acid is 100% over the six contiguous5’-most nucleotides of the target sequence of the complementary strandof the target nucleic acid. The percent complementarity between thespacer sequence and the target nucleic acid can be at least 60% overabout 20 contiguous nucleotides. The length of the spacer sequence andthe target nucleic acid can differ by 1 to 6 nucleotides, which may bethought of as a bulge or bulges.

The spacer sequence can be designed or chosen using a computer program.The computer program can use variables, such as predicted meltingtemperature, secondary structure formation, predicted annealingtemperature, sequence identity, genomic context, chromatinaccessibility, % GC, frequency of genomic occurrence (e.g., of sequencesthat are identical or are similar but vary in one or more spots as aresult of mismatch, insertion or deletion), methylation status, presenceof SNPs, and the like.

Minimum CRISPR Repeat Sequence

In some aspects, a minimum CRISPR repeat sequence is a sequence with atleast about 30%, about 40%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or 100% sequenceidentity to a reference CRISPR repeat sequence (e.g., crRNA from S.pyogenes).

In some aspects, a minimum CRISPR repeat sequence comprises nucleotidesthat can hybridize to a minimum tracrRNA sequence in a cell. The minimumCRISPR repeat sequence and a minimum tracrRNA sequence can form aduplex, i.e. a base-paired double-stranded structure. Together, theminimum CRISPR repeat sequence and the minimum tracrRNA sequence canbind to the site-directed polypeptide. At least a part of the minimumCRISPR repeat sequence can hybridize to the minimum tracrRNA sequence.At least a part of the minimum CRISPR repeat sequence can comprise atleast about 30%, about 40%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or 100%complementary to the minimum tracrRNA sequence. In some aspects, atleast a part of the minimum CRISPR repeat sequence comprises at mostabout 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about75%, about 80%, about 85%, about 90%, about 95%, or 100% complementaryto the minimum tracrRNA sequence.

The minimum CRISPR repeat sequence can have a length from about 7nucleotides to about 100 nucleotides. For example, the length of theminimum CRISPR repeat sequence is from about 7 nucleotides (nt) to about50 nt, from about 7 nt to about 40 nt, from about 7 nt to about 30 nt,from about 7 nt to about 25 nt, from about 7 nt to about 20 nt, fromabout 7 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt toabout 20 nt, from about 8 nt to about 15 nt, from about 15 nt to about100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt, orfrom about 15 nt to about 25 nt. In some aspects, the minimum CRISPRrepeat sequence is approximately 9 nucleotides in length. In someaspects, the minimum CRISPR repeat sequence is approximately 12nucleotides in length.

The minimum CRISPR repeat sequence can be at least about 60% identicalto a reference minimum CRISPR repeat sequence (e.g., wild-type crRNAfrom S. pyogenes) over a stretch of at least 6, 7, or 8 contiguousnucleotides. For example, the minimum CRISPR repeat sequence can be atleast about 65% identical, at least about 70% identical, at least about75% identical, at least about 80% identical, at least about 85%identical, at least about 90% identical, at least about 95% identical,at least about 98% identical, at least about 99% identical or 100%identical to a reference minimum CRISPR repeat sequence over a stretchof at least 6, 7, or 8 contiguous nucleotides.

Minimum tracrRNA Sequence

A minimum tracrRNA sequence can be a sequence with at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% sequence identity to areference tracrRNA sequence (e.g., wild type tracrRNA from S. pyogenes).

A minimum tracrRNA sequence can comprise nucleotides that hybridize to aminimum CRISPR repeat sequence in a cell. A minimum tracrRNA sequenceand a minimum CRISPR repeat sequence form a duplex, i.e. a base-paireddouble-stranded structure. Together, the minimum tracrRNA sequence andthe minimum CRISPR repeat can bind to a site-directed polypeptide. Atleast a part of the minimum tracrRNA sequence can hybridize to theminimum CRISPR repeat sequence. The minimum tracrRNA sequence can be atleast about 30%, about 40%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or 100%complementary to the minimum CRISPR repeat sequence.

The minimum tracrRNA sequence can have a length from about 7 nucleotidesto about 100 nucleotides. For example, the minimum tracrRNA sequence canbe from about 7 nucleotides (nt) to about 50 nt, from about 7 nt toabout 40 nt, from about 7 nt to about 30 nt, from about 7 nt to about 25nt, from about 7 nt to about 20 nt, from about 7 nt to about 15 nt, fromabout 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt toabout 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt or from about 15 nt to about 25 nt long.The minimum tracrRNA sequence can be approximately 9 nucleotides inlength. The minimum tracrRNA sequence can be approximately 12nucleotides. The minimum tracrRNA can consist of tracrRNA nt 23-48described in Jinek et al., supra.

The minimum tracrRNA sequence can be at least about 60% identical to areference minimum tracrRNA (e.g., wild type, tracrRNA from S. pyogenes)sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.For example, the minimum tracrRNA sequence can be at least about 65%identical, about 70% identical, about 75% identical, about 80%identical, about 85% identical, about 90% identical, about 95%identical, about 98% identical, about 99% identical or 100% identical toa reference minimum tracrRNA sequence over a stretch of at least 6, 7,or 8 contiguous nucleotides.

The duplex between the minimum CRISPR RNA and the minimum tracrRNA cancomprise a double helix. The duplex between the minimum CRISPR RNA andthe minimum tracrRNA can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 or more nucleotides. The duplex between the minimum CRISPR RNAand the minimum tracrRNA can comprise at most about 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more nucleotides.

The duplex can comprise a mismatch (i.e., the two strands of the duplexare not 100% complementary). The duplex can comprise at least about 1,2, 3, 4, or 5 or mismatches. The duplex can comprise at most about 1, 2,3, 4, or 5 or mismatches. The duplex can comprise no more than 2mismatches.

Bulges

In some cases, there can be a “bulge” in the duplex between the minimumCRISPR RNA and the minimum tracrRNA. A bulge is an unpaired region ofnucleotides within the duplex. A bulge can contribute to the binding ofthe duplex to the site-directed polypeptide. The bulge can comprise, onone side of the duplex, an unpaired 5’-XXXY-3’ where X is any purine andY comprises a nucleotide that can form a wobble pair with a nucleotideon the opposite strand, and an unpaired nucleotide region on the otherside of the duplex. The number of unpaired nucleotides on the two sidesof the duplex can be different.

In one example, the bulge can comprise an unpaired purine (e.g.,adenine) on the minimum CRISPR repeat strand of the bulge. In someexamples, the bulge can comprise an unpaired 5’-AAGY-3’ of the minimumtracrRNA sequence strand of the bulge, where Y comprises a nucleotidethat can form a wobble pairing with a nucleotide on the minimum CRISPRrepeat strand.

A bulge on the minimum CRISPR repeat side of the duplex can comprise atleast 1, 2, 3, 4, or 5 or more unpaired nucleotides. A bulge on theminimum CRISPR repeat side of the duplex can comprise at most 1, 2, 3,4, or 5 or more unpaired nucleotides. A bulge on the minimum CRISPRrepeat side of the duplex can comprise 1 unpaired nucleotide.

A bulge on the minimum tracrRNA sequence side of the duplex can compriseat least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides.A bulge on the minimum tracrRNA sequence side of the duplex can compriseat most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. Abulge on a second side of the duplex (e.g., the minimum tracrRNAsequence side of the duplex) can comprise 4 unpaired nucleotides.

A bulge can comprise at least one wobble pairing. In some examples, abulge can comprise at most one wobble pairing. A bulge can comprise atleast one purine nucleotide. A bulge can comprise at least 3 purinenucleotides. A bulge sequence can comprise at least 5 purinenucleotides. A bulge sequence can comprise at least one guaninenucleotide. In some examples, a bulge sequence can comprise at least oneadenine nucleotide.

Hairpins

In various examples, one or more hairpins can be located 3’ to theminimum tracrRNA in the 3’ tracrRNA sequence.

The hairpin can start at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,or 20 or more nucleotides 3’ from the last paired nucleotide in theminimum CRISPR repeat and minimum tracrRNA sequence duplex. The hairpincan start at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or morenucleotides 3’ of the last paired nucleotide in the minimum CRISPRrepeat and minimum tracrRNA sequence duplex.

The hairpin can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, or 20 or more consecutive nucleotides. The hairpin can comprise atmost about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more consecutivenucleotides.

The hairpin can comprise a CC dinucleotide (i.e., two consecutivecytosine nucleotides).

The hairpin can comprise duplexed nucleotides (e.g., nucleotides in ahairpin, hybridized together). For example, a hairpin can comprise a CCdinucleotide that is hybridized to a GG dinucleotide in a hairpin duplexof the 3’ tracrRNA sequence.

One or more of the hairpins can interact with guide RNA-interactingregions of a site-directed polypeptide.

In some examples, there are two or more hairpins, and in other examplesthere are three or more hairpins.

3’ tracrRNA Sequence

A 3’ tracrRNA sequence can comprise a sequence with at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% sequence identity to areference tracrRNA sequence (e.g., a tracrRNA from S. pyogenes).

The 3’ tracrRNA sequence can have a length from about 6 nucleotides toabout 100 nucleotides. For example, the 3’ tracrRNA sequence can have alength from about 6 nucleotides (nt) to about 50 nt, from about 6 nt toabout 40 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25nt, from about 6 nt to about 20 nt, from about 6 nt to about 15 nt, fromabout 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt toabout 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt, or from about 15 nt to about 25 nt. The3’ tracrRNA sequence can have a length of approximately 14 nucleotides.

The 3’ tracrRNA sequence can be at least about 60% identical to areference 3’ tracrRNA sequence (e.g., wild type 3’ tracrRNA sequencefrom S. pyogenes) over a stretch of at least 6, 7, or 8 contiguousnucleotides. For example, the 3’ tracrRNA sequence can be at least about60% identical, about 65% identical, about 70% identical, about 75%identical, about 80% identical, about 85% identical, about 90%identical, about 95% identical, about 98% identical, about 99%identical, or 100% identical, to a reference 3’ tracrRNA sequence (e.g.,wild type 3’ tracrRNA sequence from S. pyogenes) over a stretch of atleast 6, 7, or 8 contiguous nucleotides.

The 3’ tracrRNA sequence can comprise more than one duplexed region(e.g., hairpin, hybridized region). The 3’ tracrRNA sequence cancomprise two duplexed regions.

The 3’ tracrRNA sequence can comprise a stem loop structure. The stemloop structure in the 3’ tracrRNA can comprise at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15 or 20 or more nucleotides. The stem loop structure inthe 3’ tracrRNA can comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ormore nucleotides. The stem loop structure can comprise a functionalmoiety. For example, the stem loop structure can comprise an aptamer, aribozyme, a protein-interacting hairpin, a CRISPR array, an intron, oran exon. The stem loop structure can comprise at least about 1, 2, 3, 4,or 5 or more functional moieties. The stem loop structure can compriseat most about 1, 2, 3, 4, or 5 or more functional moieties.

The hairpin in the 3’ tracrRNA sequence can comprise a P-domain. In someexamples, the P-domain can comprise a double-stranded region in thehairpin.

tracrRNA Extension Sequence

A tracrRNA extension sequence may be provided whether the tracrRNA is inthe context of single-molecule guides or double-molecule guides. ThetracrRNA extension sequence can have a length from about 1 nucleotide toabout 400 nucleotides. The tracrRNA extension sequence can have a lengthof more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360,380, or 400 nucleotides. The tracrRNA extension sequence can have alength from about 20 to about 5000 or more nucleotides. The tracrRNAextension sequence can have a length of more than 1000 nucleotides. ThetracrRNA extension sequence can have a length of less than 1, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 or morenucleotides. The tracrRNA extension sequence can have a length of lessthan 1000 nucleotides. The tracrRNA extension sequence can comprise lessthan 10 nucleotides in length. The tracrRNA extension sequence can be10-30 nucleotides in length. The tracrRNA extension sequence can be30-70 nucleotides in length.

The tracrRNA extension sequence can comprise a functional moiety (e.g.,a stability control sequence, ribozyme, endoribonuclease bindingsequence). The functional moiety can comprise a transcriptionalterminator segment (i.e., a transcription termination sequence). Thefunctional moiety can have a total length from about 10 nucleotides (nt)to about 100 nucleotides, from about 10 nt to about 20 nt, from about 20nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 nt toabout 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt,or from about 90 nt to about 100 nt, from about 15 nt to about 80 nt,from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, fromabout 15 nt to about 30 nt, or from about 15 nt to about 25 nt. Thefunctional moiety can function in a eukaryotic cell. The functionalmoiety can function in a prokaryotic cell. The functional moiety canfunction in both eukaryotic and prokaryotic cells.

Non-limiting examples of suitable tracrRNA extension functional moietiesinclude a 3’ poly-adenylated tail, a riboswitch sequence (e.g., to allowfor regulated stability and/or regulated accessibility by proteins andprotein complexes), a sequence that forms a dsRNA duplex (i.e., ahairpin), a sequence that targets the RNA to a subcellular location(e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like). The tracrRNA extension sequence cancomprise a primer binding site or a molecular index (e.g., barcodesequence). The tracrRNA extension sequence can comprise one or moreaffinity tags.

Single-Molecule Guide Linker Sequence

The linker sequence of a single-molecule guide nucleic acid can have alength from about 3 nucleotides to about 100 nucleotides. In Jinek etal., supra, for example, a simple 4 nucleotide “tetraloop” (-GAAA-) wasused, Science, 337(6096):816-821 (2012). An illustrative linker has alength from about 3 nucleotides (nt) to about 90 nt, from about 3 nt toabout 80 nt, from about 3 nt to about 70 nt, from about 3 nt to about 60nt, from about 3 nt to about 50 nt, from about 3 nt to about 40 nt, fromabout 3 nt to about 30 nt, from about 3 nt to about 20 nt, from about 3nt to about 10 nt. For example, the linker can have a length from about3 nt to about 5 nt, from about 5 nt to about 10 nt, from about 10 nt toabout 15 nt, from about 15 nt to about 20 nt, from about 20 nt to about25 nt, from about 25 nt to about 30 nt, from about 30 nt to about 35 nt,from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, fromabout 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90nt to about 100 nt. The linker of a single-molecule guide nucleic acidcan be between 4 and 40 nucleotides. The linker can be at least about100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, or 7000 or more nucleotides. The linker can be at most about100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, or 7000 or more nucleotides.

Linkers can comprise any of a variety of sequences, although in someexamples the linker will not comprise sequences that have extensiveregions of homology with other portions of the guide RNA, which mightcause intramolecular binding that could interfere with other functionalregions of the guide. In Jinek et al., supra, a simple 4 nucleotidesequence -GAAA-was used, Science, 337(6096):816-821 (2012), but numerousother sequences, including longer sequences can likewise be used.

The linker sequence can comprise a functional moiety. For example, thelinker sequence can comprise one or more features, including an aptamer,a ribozyme, a protein-interacting hairpin, a protein binding site, aCRISPR array, an intron, or an exon. The linker sequence can comprise atleast about 1, 2, 3, 4, or 5 or more functional moieties. In someexamples, the linker sequence can comprise at most about 1, 2, 3, 4, or5 or more functional moieties.

Nucleic Acid Modifications (Chemical and Structural Modifications)

In some aspects, polynucleotides introduced into cells can comprise oneor more modifications that can be used individually or in combination,for example, to enhance activity, stability or specificity, alterdelivery, reduce innate immune responses in host cells, or for otherenhancements, as further described herein and known in the art.

In certain examples, modified polynucleotides can be used in theCRISPR/Cas9 or CRISPR/Cpfl system, in which case the guide RNAs (eithersingle-molecule guides or double-molecule guides) and/or a DNA or an RNAencoding a Cas9 or Cpfl endonuclease introduced into a cell can bemodified, as described and illustrated below. Such modifiedpolynucleotides can be used in the CRISPR/Cas9 or CRISPR/Cpfl system toedit any one or more genomic loci.

Using the CRISPR/Cas9 or CRISPR/Cpfl system for purposes of non-limitingillustrations of such uses, modifications of guide RNAs can be used toenhance the formation or stability of the CRISPR/Cas9 or CRISPR/Cpflgenome editing complex comprising guide RNAs, which can besingle-molecule guides or double-molecule, and a Cas9 or Cpflendonuclease. Modifications of guide RNAs can also or alternatively beused to enhance the initiation, stability or kinetics of interactionsbetween the genome editing complex with the target sequence in thegenome, which can be used, for example, to enhance on-target activity.Modifications of guide RNAs can also or alternatively be used to enhancespecificity, e.g., the relative rates of genome editing at the on-targetsite as compared to effects at other (off-target) sites.

Modifications can also or alternatively be used to increase thestability of a guide RNA, e.g., by increasing its resistance todegradation by ribonucleases (RNases) present in a cell, thereby causingits half-life in the cell to be increased. Modifications enhancing guideRNA half-life can be particularly useful in aspects in which a Cas orCpfl endonuclease is introduced into the cell to be edited via an RNAthat needs to be translated in order to generate endonuclease, becauseincreasing the half-life of guide RNAs introduced at the same time asthe RNA encoding the endonuclease can be used to increase the time thatthe guide RNAs and the encoded Cas or Cpfl endonuclease co-exist in thecell.

Modifications can also or alternatively be used to decrease thelikelihood or degree to which RNAs introduced into cells elicit innateimmune responses. Such responses, which have been well characterized inthe context of RNA interference (RNAi), including small-interfering RNAs(siRNAs), as described below and in the art, tend to be associated withreduced half-life of the RNA and/or the elicitation of cytokines orother factors associated with immune responses.

One or more types of modifications can also be made to RNAs encoding anendonuclease that are introduced into a cell, including, withoutlimitation, modifications that enhance the stability of the RNA (such asby increasing its degradation by RNases present in the cell),modifications that enhance translation of the resulting product (i.e.the endonuclease), and/or modifications that decrease the likelihood ordegree to which the RNAs introduced into cells elicit innate immuneresponses.

Combinations of modifications, such as the foregoing and others, canlikewise be used. In the case of CRISPR/Cas9 or CRISPR/Cpfl, forexample, one or more types of modifications can be made to guide RNAs(including those exemplified above), and/or one or more types ofmodifications can be made to RNAs encoding Cas endonuclease (includingthose exemplified above).

By way of illustration, guide RNAs used in the CRISPR/Cas9 orCRISPR/Cpfl system, or other smaller RNAs can be readily synthesized bychemical means, enabling a number of modifications to be readilyincorporated, as illustrated below and described in the art. Whilechemical synthetic procedures are continually expanding, purificationsof such RNAs by procedures such as high-performance liquidchromatography (HPLC, which avoids the use of gels such as PAGE) tendsto become more challenging as polynucleotide lengths increasesignificantly beyond a hundred or so nucleotides. One approach that canbe used for generating chemically-modified RNAs of greater length is toproduce two or more molecules that are ligated together. Much longerRNAs, such as those encoding a Cas9 endonuclease, are more readilygenerated enzymatically. While fewer types of modifications areavailable for use in enzymatically produced RNAs, there are stillmodifications that can be used to, e.g., enhance stability, reduce thelikelihood or degree of innate immune response, and/or enhance otherattributes, as described further below and in the art; and new types ofmodifications are regularly being developed.

By way of illustration of various types of modifications, especiallythose used frequently with smaller chemically synthesized RNAs,modifications can comprise one or more nucleotides modified at the 2’position of the sugar, in some aspects a 2’-O-alkyl, 2’-O-alkyl-O-alkyl,or 2’-fluoro-modified nucleotide. In some examples, RNA modificationsinclude 2’-fluoro, 2’-amino or 2’-O-methyl modifications on the riboseof pyrimidines, abasic residues, or an inverted base at the 3’ end ofthe RNA. Such modifications are routinely incorporated intooligonucleotides and these oligonucleotides have been shown to have ahigher Tm (i.e., higher target binding affinity) than2’-deoxyoligonucleotides against a given target.

A number of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligonucleotide; these modifiedoligos survive intact for a longer time than unmodifiedoligonucleotides. Specific examples of modified oligonucleotides includethose comprising modified backbones, for example, phosphorothioates,phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. Some oligonucleotides are oligonucleotides withphosphorothioate backbones and those with heteroatom backbones,particularly CH₂—NH—O—CH₂, CH,~N(CH₃)~O~CH₂ (known as amethylene(methylimino) or MMI backbone), CH₂—O—N (CH₃)—CH₂, CH₂ —N(CH₃)—N (CH₃)—CH₂ and O—N (CH₃)— CH₂ —CH₂ backbones, wherein the nativephosphodiester backbone is represented as O- P- O- CH,); amide backbones[see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995)];morpholino backbone structures (see Summerton and Weller, U.S. Pat. No.5,034,506); peptide nucleic acid (PNA) backbone (wherein thephosphodiester backbone of the oligonucleotide is replaced with apolyamide backbone, the nucleotides being bound directly or indirectlyto the aza nitrogen atoms of the polyamide backbone, see Nielsen et al.,Science 1991, 254, 1497). Phosphorus-containing linkages include, butare not limited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3’alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3’-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3’-5’linkages, 2’-5’ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3’-5’to 5’-3’ or 2’-5’ to 5’-2’; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Braasch and DavidCorey, Biochemistry, 41(14): 4503-4510 (2002); Genesis, Volume 30, Issue3, (2001); Heasman, Dev. Biol., 243: 209-214 (2002); Nasevicius et al.,Nat. Genet., 26:216-220 (2000); Lacerra et al., Proc. Natl. Acad. Sci.,97: 9591-9596 (2000); and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc., 122: 8595-8602 (2000).

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S, and CH₂ component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439.

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2’ position: OH, SH, SCH₃, F, OCN, OCH₃ OCH₃,OCH₃ O(CH₂)n CH₃, O(CH₂)n NH₂, or O(CH₂)n CH₃, where n is from 1 toabout 10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O—, S—, or N—alkyl; O—, S—,or N—alkenyl; SOCH₃; SO₂ CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. In some aspects, amodification includes 2’-methoxyethoxy (2’-O-CH₂CH₂OCH₃, also known as2’-O-(2-methoxyethyl)) (Martin et al, HeIv. Chim. Acta, 1995, 78, 486).Other modifications include 2’-methoxy (2’-O-CH₃), 2’-propoxy (2’-OCH₂CH₂CH₃) and 2’-fluoro (2’-F). Similar modifications may also be made atother positions on the oligonucleotide, particularly the 3’ position ofthe sugar on the 3’ terminal nucleotide and the 5’ position of 5’terminal nucleotide. Oligonucleotides may also have sugar mimetics, suchas cyclobutyls in place of the pentofuranosyl group.

In some examples, both a sugar and an internucleoside linkage, i.e., thebackbone, of the nucleotide units can be replaced with novel groups. Thebase units can be maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, anoligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide can bereplaced with an amide containing backbone, for example, anaminoethylglycine backbone. The nucleobases can be retained and bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative U.S. patents that teach the preparation of PNAcompounds comprise, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262. Further teaching of PNA compounds can be foundin Nielsen et al, Science, 254: 1497-1500 (1991).

Guide RNAs can also include, additionally or alternatively, nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude adenine (A), guanine (G), thymine (T), cytosine (C), and uracil(U). Modified nucleobases include nucleobases found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2’ deoxycytosine and often referred to in theart as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine,2-(methylamino) adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil,2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine,7-deazaguanine, N6 (6-aminohexyl)adenine, and 2,6-diaminopurine.Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, pp75-77 (1980); Gebeyehu et al., Nucl. Acids Res. 15:4513 (1997). A“universal” base known in the art, e.g., inosine, can also be included.5-Me-C substitutions have been shown to increase nucleic acid duplexstability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu,B., eds., Antisense Research and Applications, CRC Press, Boca Raton,1993, pp. 276-278) and are aspects of base substitutions.

Modified nucleobases can comprise other synthetic and naturalnucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5- bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and3-deazaadenine.

Further, nucleobases can comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience And Engineering’, pages 858-859, Kroschwitz, J.I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandleChemie, International Edition’, 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, Antisense Research and Applications’,pages 289- 302, Crooke, S.T. and Lebleu, B. ea., CRC Press, 1993.Certain of these nucleobases are particularly useful for increasing thebinding affinity of the oligomeric compounds of the present disclosure.These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6and 0-6 substituted purines, comprising 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.(Sanghvi, Y.S., Crooke, S.T. and Lebleu, B., eds, ‘Antisense Researchand Applications’, CRC Press, Boca Raton, 1993, pp. 276-278) and areaspects of base substitutions, even more particularly when combined with2’-O-methoxyethyl sugar modifications. Modified nucleobases aredescribed in U.S. Pat. Nos. 3,687,808, as well as 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091;5,614,617; 5,681,941; 5,750,692; 5,763,588; 5,830,653; 6,005,096; andU.S. Pat. Application Publication 2003/0158403.

Thus, the term “modified” refers to a non-natural sugar, phosphate, orbase that is incorporated into a guide RNA, an endonuclease, or both aguide RNA and an endonuclease. It is not necessary for all positions ina given oligonucleotide to be uniformly modified, and in fact more thanone of the aforementioned modifications can be incorporated in a singleoligonucleotide, or even in a single nucleoside within anoligonucleotide.

The guide RNAs and/or mRNA (or DNA) encoding an endonuclease can bechemically linked to one or more moieties or conjugates that enhance theactivity, cellular distribution, or cellular uptake of theoligonucleotide. Such moieties comprise, but are not limited to, lipidmoieties such as a cholesterol moiety [Letsinger et al., Proc. Natl.Acad. Sci. USA, 86: 6553-6556 (1989)]; cholic acid [Manoharan et al.,Bioorg. Med. Chem. Let., 4: 1053-1060 (1994)]; a thioether, e.g.,hexyl-S- tritylthiol [Manoharan et al, Ann. N. Y. Acad. Sci., 660:306-309 (1992) and Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-2770 (1993)]; a thiocholesterol [Oberhauser et al., Nucl. AcidsRes., 20: 533-538 (1992)]; an aliphatic chain, e.g., dodecandiol orundecyl residues [Kabanov et al., FEBS Lett., 259: 327-330 (1990) andSvinarchuk et al., Biochimie, 75: 49- 54 (1993)]; a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al., Tetrahedron Lett., 36:3651-3654 (1995) and Shea et al., Nucl. Acids Res., 18: 3777-3783(1990)]; a polyamine or a polyethylene glycol chain [Mancharan et al.,Nucleosides & Nucleotides, 14: 969-973 (1995)]; adamantane acetic acid[Manoharan et al., Tetrahedron Lett., 36: 3651-3654 (1995)]; a palmitylmoiety [(Mishra et al., Biochim. Biophys. Acta, 1264: 229-237 (1995)];or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety[Crooke et al., J. Pharmacol. Exp. Ther., 277: 923-937 (1996)]. See alsoU.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599, 928 and 5,688,941.

Sugars and other moieties can be used to target proteins and complexescomprising nucleotides, such as cationic polysomes and liposomes, toparticular sites. For example, hepatic cell directed transfer can bemediated via asialoglycoprotein receptors (ASGPRs); see, e.g., Hu, etal., Protein Pept Lett. 21(10):1025-30 (2014). Other systems known inthe art and regularly developed can be used to target biomolecules ofuse in the present case and/or complexes thereof to particular targetcells of interest.

These targeting moieties or conjugates can include conjugate groupscovalently bound to functional groups, such as primary or secondaryhydroxyl groups. Conjugate groups of the present disclosure includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of this presentdisclosure, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this present disclosure, include groups that improveuptake, distribution, metabolism or excretion of the compounds of thepresent disclosure. Representative conjugate groups are disclosed inInternational Patent Application No. PCT/US92/09196, filed Oct. 23, 1992(published as WO1993007883), and U.S. Pat. No. 6,287,860. Conjugatemoieties include, but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac- glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or adamantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941.

Longer polynucleotides that are less amenable to chemical synthesis andare typically produced by enzymatic synthesis can also be modified byvarious means. Such modifications can include, for example, theintroduction of certain nucleotide analogs, the incorporation ofparticular sequences or other moieties at the 5’ or 3’ ends ofmolecules, and other modifications. By way of illustration, the mRNAencoding Cas9 is approximately 4 kb in length and can be synthesized byin vitro transcription. Modifications to the mRNA can be applied to,e.g., increase its translation or stability (such as by increasing itsresistance to degradation with a cell), or to reduce the tendency of theRNA to elicit an innate immune response that is often observed in cellsfollowing introduction of exogenous RNAs, particularly longer RNAs suchas that encoding Cas9.

Numerous such modifications have been described in the art, such aspolyA tails, 5’ cap analogs (e.g., Anti Reverse Cap Analog (ARCA) orm7G(5’)ppp(5’)G (mCAP)), modified 5’ or 3’ untranslated regions (UTRs),use of modified bases (such as Pseudo-UTP, 2-Thio-UTP,5-Methylcytidine-5’-Triphosphate (5-Methyl-CTP) or N6-Methyl-ATP), ortreatment with phosphatase to remove 5’ terminal phosphates. These andother modifications are known in the art, and new modifications of RNAsare regularly being developed.

There are numerous commercial suppliers of modified RNAs, including forexample, TriLink Biotech, AxoLabs, Bio-Synthesis Inc., Dharmacon andmany others. As described by TriLink, for example, 5-Methyl-CTP can beused to impart desirable characteristics, such as increased nucleasestability, increased translation or reduced interaction of innate immunereceptors with in vitro transcribed RNA.5-Methylcytidine-5’-Triphosphate (5-Methyl-CTP), N6-Methyl-ATP, as wellas Pseudo-UTP and 2-Thio-UTP, have also been shown to reduce innateimmune stimulation in culture and in vivo while enhancing translation,as illustrated in publications by Kormann et al. and Warren et al.referred to below.

It has been shown that chemically modified mRNA delivered in vivo can beused to achieve improved therapeutic effects; see, e.g., Kormann et al.,Nature Biotechnology 29, 154-157 (2011). Such modifications can be used,for example, to increase the stability of the RNA molecule and/or reduceits immunogenicity. Using chemical modifications such as Pseudo-U,N6-Methyl-A, 2-Thio-U and 5-Methyl-C, it was found that substitutingjust one quarter of the uridine and cytidine residues with 2-Thio-U and5-Methyl-C respectively resulted in a significant decrease in toll-likereceptor (TLR) mediated recognition of the mRNA in mice. By reducing theactivation of the innate immune system, these modifications can be usedto effectively increase the stability and longevity of the mRNA in vivo;see, e.g., Kormann et al., supra.

It has also been shown that repeated administration of syntheticmessenger RNAs incorporating modifications designed to bypass innateanti-viral responses can reprogram differentiated human cells topluripotency. See, e.g., Warren, et al., Cell Stem Cell, 7(5):618-30(2010). Such modified mRNAs that act as primary reprogramming proteinscan be an efficient means of reprogramming multiple human cell types.Such cells are referred to as induced pluripotency stem cells (iPSCs),and it was found that enzymatically synthesized RNA incorporating5-Methyl-CTP, Pseudo-UTP and an Anti Reverse Cap Analog (ARCA) could beused to effectively evade the cell’s antiviral response; see, e.g.,Warren et al., supra.

Other modifications of polynucleotides described in the art include, forexample, the use of polyA tails, the addition of 5’ cap analogs (such asm7G(5’)ppp(5’)G (mCAP)), modifications of 5’ or 3’ untranslated regions(UTRs), or treatment with phosphatase to remove 5’ terminal phosphates -and new approaches are regularly being developed.

A number of compositions and techniques applicable to the generation ofmodified RNAs for use herein have been developed in connection with themodification of RNA interference (RNAi), including small-interferingRNAs (siRNAs). siRNAs present particular challenges in vivo becausetheir effects on gene silencing via mRNA interference are generallytransient, which can require repeat administration. In addition, siRNAsare double-stranded RNAs (dsRNA) and mammalian cells have immuneresponses that have evolved to detect and neutralize dsRNA, which isoften a by-product of viral infection. Thus, there are mammalian enzymessuch as PKR (dsRNA-responsive kinase), and potentially retinoicacid-inducible gene I (RIG-I), that can mediate cellular responses todsRNA, as well as Toll-like receptors (such as TLR3, TLR7 and TLR8) thatcan trigger the induction of cytokines in response to such molecules;see, e.g., the reviews by Angart et al., Pharmaceuticals (Basel) 6(4):440-468 (2013); Kanasty et al., Molecular Therapy 20(3): 513-524 (2012);Burnett et al., Biotechnol J. 6(9):1130-46 (2011); Judge and MacLachlan,Hum Gene Ther 19(2):111-24 (2008); and references cited therein.

A large variety of modifications have been developed and applied toenhance RNA stability, reduce innate immune responses, and/or achieveother benefits that can be useful in connection with the introduction ofpolynucleotides into human cells, as described herein; see, e.g., thereviews by Whitehead KA et al., Annual Review of Chemical andBiomolecular Engineering, 2: 77-96 (2011); Gaglione and Messere, MiniRev Med Chem, 10(7):578-95 (2010); Chernolovskaya et al, Curr Opin MolTher., 12(2):158-67 (2010); Deleavey et al., Curr Protoc Nucleic AcidChem Chapter 16:Unit 16.3 (2009); Behlke, Oligonucleotides 18(4):305-19(2008); Fucini et al., Nucleic Acid Ther 22(3): 205-210 (2012); Bremsenet al., Front Genet 3:154 (2012).

As noted above, there are a number of commercial suppliers of modifiedRNAs, many of which have specialized in modifications designed toimprove the effectiveness of siRNAs. A variety of approaches are offeredbased on various findings reported in the literature. For example,Dharmacon notes that replacement of a non-bridging oxygen with sulfur(phosphorothioate, PS) has been extensively used to improve nucleaseresistance of siRNAs, as reported by Kole, Nature Reviews Drug Discovery11:125-140 (2012). Modifications of the 2’-position of the ribose havebeen reported to improve nuclease resistance of the internucleotidephosphate bond while increasing duplex stability (Tm), which has alsobeen shown to provide protection from immune activation. A combinationof moderate PS backbone modifications with small, well-tolerated2’-substitutions (2’-O-Methyl, 2’-Fluoro, 2’-Hydro) have been associatedwith highly stable siRNAs for applications in vivo, as reported bySoutschek et al. Nature 432:173-178 (2004); and 2’-O-Methylmodifications have been reported to be effective in improving stabilityas reported by Volkov, Oligonucleotides 19:191-202 (2009). With respectto decreasing the induction of innate immune responses, modifyingspecific sequences with 2’-O-Methyl, 2’-Fluoro, 2’-Hydro have beenreported to reduce TLR7/TLR8 interaction while generally preservingsilencing activity; see, e.g., Judge et al., Mol. Ther. 13 :494-505(2006); and Cekaite et al., J. Mol. Biol. 365:90-108 (2007). Additionalmodifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine,5-methyluracil, and N6-methyladenosine have also been shown to minimizethe immune effects mediated by TLR3, TLR7, and TLR8; see, e.g., Kariko,K. et al., Immunity 23:165-175 (2005).

As is also known in the art, and commercially available, a number ofconjugates can be applied to polynucleotides, such as RNAs, for useherein that can enhance their delivery and/or uptake by cells, includingfor example, cholesterol, tocopherol and folic acid, lipids, peptides,polymers, linkers and aptamers; see, e.g., the review by Winkler, Ther.Deliv. 4:791-809 (2013), and references cited therein.

Codon-Optimization

A polynucleotide encoding a site-directed polypeptide can becodon-optimized according to methods standard in the art for expressionin the cell containing the target DNA of interest. For example, if theintended target nucleic acid is in a human cell, a human codon-optimizedpolynucleotide encoding Cas9 is contemplated for use for producing theCas9 polypeptide.

Complexes of a Genome-Targeting Nucleic Acid and a Site-DirectedPolypeptide

A genome-targeting nucleic acid interacts with a site-directedpolypeptide (e.g., a nucleic acid-guided nuclease such as Cas9), therebyforming a complex. The genome-targeting nucleic acid guides thesite-directed polypeptide to a target nucleic acid.

Ribonucleoprotein Complexes (RNPs)

The site-directed polypeptide and genome-targeting nucleic acid can eachbe administered separately to a cell or a patient. On the other hand,the site-directed polypeptide can be pre-complexed with one or moreguide RNAs, or one or more crRNA together with a tracrRNA. Thepre-complexed material can then be administered to a cell or a patient.Such pre-complexed material is known as a ribonucleoprotein particle(RNP). The site-directed polypeptide in the RNP can be, for example, aCas9 endonuclease or a Cpfl endonuclease. The site-directed polypeptidecan be flanked at the N-terminus, the C-terminus, or both the N-terminusand C-terminus by one or more nuclear localization signals (NLSs). Forexample, a Cas9 endonuclease can be flanked by two NLSs, one NLS locatedat the N-terminus and the second NLS located at the C-terminus. The NLScan be any NLS known in the art, such as a SV40 NLS. The weight ratio ofgenome-targeting nucleic acid to site-directed polypeptide in the RNPcan be 1:1. For example, the weight ratio of sgRNA to Cas9 endonucleasein the RNP can be 1:1.

Nucleic Acids Encoding System Components

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a genome-targeting nucleic acid of the disclosure, asite-directed polypeptide of the disclosure, and/or any nucleic acid orproteinaceous molecule necessary to carry out the aspects of the methodsof the disclosure.

The nucleic acid encoding a genome-targeting nucleic acid of thedisclosure, a site-directed polypeptide of the disclosure, and/or anynucleic acid or proteinaceous molecule necessary to carry out theaspects of the methods of the disclosure can comprise a vector (e.g., arecombinant expression vector).

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double-stranded DNAloop into which additional nucleic acid segments can be ligated. Anothertype of vector is a viral vector, wherein additional nucleic acidsegments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome.

In some examples, vectors can be capable of directing the expression ofnucleic acids to which they are operatively linked. Such vectors arereferred to herein as “recombinant expression vectors”, or more simply“expression vectors”, which serve equivalent functions.

The term “operably linked” means that the nucleotide sequence ofinterest is linked to regulatory sequence(s) in a manner that allows forexpression of the nucleotide sequence. The term “regulatory sequence” isintended to include, for example, promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are well known in the art and are described, forexample, in Goeddel; Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, CA (1990). Regulatory sequences includethose that direct constitutive expression of a nucleotide sequence inmany types of host cells, and those that direct expression of thenucleotide sequence only in certain host cells (e.g., tissue-specificregulatory sequences). It will be appreciated by those skilled in theart that the design of the expression vector can depend on such factorsas the choice of the target cell, the level of expression desired, andthe like.

Expression vectors contemplated include, but are not limited to, viralvectors based on vaccinia virus, poliovirus, adenovirus,adeno-associated virus, SV40, herpes simplex virus, humanimmunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleennecrosis virus, and vectors derived from retroviruses such as RousSarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus,human immunodeficiency virus, myeloproliferative sarcoma virus, andmammary tumor virus) and other recombinant vectors. Other vectorscontemplated for eukaryotic target cells include, but are not limitedto, the vectors pXTl, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).Other vectors can be used so long as they are compatible with the hostcell.

In some examples, a vector can comprise one or more transcription and/ortranslation control elements. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationcontrol elements, including constitutive and inducible promoters,transcription enhancer elements, transcription terminators, etc. can beused in the expression vector. The vector can be a self-inactivatingvector that either inactivates the viral sequences or the components ofthe CRISPR machinery or other elements.

Non-limiting examples of suitable eukaryotic promoters (i.e., promotersfunctional in a eukaryotic cell) include those from cytomegalovirus(CMV) immediate early, herpes simplex virus (HSV) thymidine kinase,early and late SV40, long terminal repeats (LTRs) from retrovirus, humanelongation factor-1 promoter (EF1), a hybrid construct comprising thecytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter(CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1locus promoter (PGK), and mouse metallothionein-I.

For expressing small RNAs, including guide RNAs used in connection withCas endonuclease, various promoters such as RNA polymerase IIIpromoters, including for example U6 and Hl, can be advantageous.Descriptions of and parameters for enhancing the use of such promotersare known in art, and additional information and approaches areregularly being described; see, e.g., Ma, H. et al., Molecular Therapy -Nucleic Acids 3, e161 (2014) doi:10.1038/mtna.2014.12.

The expression vector can also contain a ribosome binding site fortranslation initiation and a transcription terminator. The expressionvector can also comprise appropriate sequences for amplifyingexpression. The expression vector can also include nucleotide sequencesencoding non-native tags (e.g., histidine tag, hemagglutinin tag, greenfluorescent protein, etc.) that are fused to the site-directedpolypeptide, thus resulting in a fusion protein.

A promoter can be an inducible promoter (e.g., a heat shock promoter,tetracycline-regulated promoter, steroid-regulated promoter,metal-regulated promoter, estrogen receptor-regulated promoter, etc.).The promoter can be a constitutive promoter (e.g., CMV promoter, UBCpromoter). In some cases, the promoter can be a spatially restrictedand/or temporally restricted promoter (e.g., a tissue specific promoter,a cell type specific promoter, etc.).

The nucleic acid encoding a genome-targeting nucleic acid of thedisclosure and/or a site-directed polypeptide can be packaged into or onthe surface of delivery vehicles for delivery to cells. Deliveryvehicles contemplated include, but are not limited to, nanospheres,liposomes, quantum dots, nanoparticles, polyethylene glycol particles,hydrogels, and micelles. As described in the art, a variety of targetingmoieties can be used to enhance the preferential interaction of suchvehicles with desired cell types or locations.

Introduction of the complexes, polypeptides, and nucleic acids of thedisclosure into cells can occur by viral or bacteriophage infection,transfection, conjugation, protoplast fusion, lipofection,electroporation, nucleofection, calcium phosphate precipitation,polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology,calcium phosphate precipitation, direct micro-injection,nanoparticle-mediated nucleic acid delivery, and the like.

Therapeutic Approach

Provided herein are methods for treating a patient with MyotonicDystrophy Type 1. Myotonic Dystrophy Type 1 is caused by abnormalexpansion of a trinucleotide CTG repeat in the 3’ untranslated region(UTR) of the DMPK gene. In most people, the CTG segment is repeatedfewer than 34 times. In patients with Myotonic Dystrophy Type 1, thenumber of CTG repeats can range from 50 to 5,000 repeats. This numbercan be different from one patient to another.

The term “trinucleotide repeat expansion” means a series of three bases(for example, CTG) repeated at least twice. In certain examples, thetrinucleotide repeat expansion may be located in the 3’ untranslatedregion (UTR) of a DMPK nucleic acid. In certain examples, a pathogenictrinucleotide repeat expansion includes at least 50 repeats of CTG in aDMPK nucleic acid and is associated with disease. In other examples, apathogenic trinucleotide repeat expansion includes at least 51, 52, 53,54, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 500, 800, 1000, 3000,5000 or more repeats. In certain examples, the repeats are consecutive.In certain examples, the repeats are interrupted by one or morenucleobases. In certain examples, a wild-type trinucleotide repeatexpansion includes 34 or fewer repeats of CTG in a DMPK nucleic acid. Inother examples, a wild-type trinucleotide repeat expansion includes 33,32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 repeat. In certainexamples, the entire trinucleotide repeat expansion is deleted. In otherexamples, a portion of the trinucleotide repeat expansion is deleted.

An aspect of such method is an ex vivo cell-based therapy. For example,a biopsy of the patient’s skeletal muscle is performed. Then, myocytesare isolated from the biopsied material. Then, the chromosomal DNA ofthe myocytes can be edited using the materials and methods describedherein. Finally, the edited myocytes are implanted into the patient. Anysource or type of cell may be used as the progenitor cell.

Another aspect of such method is an ex vivo cell-based therapy. Forexample, a biopsy of the patient’s smooth muscle or cardiac muscle isperformed. Then, myocytes are isolated from the biopsied material. Then,the chromosomal DNA of the myocytes can be edited using the materialsand methods described herein. Finally, the edited myocytes are implantedinto the patient. Any source or type of cell may be used as theprogenitor cell.

Another aspect of such method is an ex vivo cell-based therapy. Forexample, a patient specific induced pluripotent stem cell (iPSC) can becreated. Then, the chromosomal DNA of these iPS cells can be editedusing the materials and methods described herein. Next, thegenome-edited iPSCs can be differentiated into other cells, e.g.,myocytes or cells of the central nervous system. Finally, thedifferentiated cells are implanted into the patient.

Yet another aspect of such method is an ex vivo cell-based therapy. Forexample, a mesenchymal stem cell can be isolated from the patient, whichcan be isolated from the patient’s bone marrow or peripheral blood.Next, the chromosomal DNA of these mesenchymal stem cells can be editedusing the materials and methods described herein. Next, thegenome-edited mesenchymal stem cells can be differentiated into othercells, e.g., myocytes or cells of the central nervous system. Finally,the differentiated cells, e.g., myocytes or cells of the central nervoussystem, are implanted into the patient.

One advantage of an ex vivo cell therapy approach is the ability toconduct a comprehensive analysis of the therapeutic prior toadministration. Nuclease-based therapeutics can have some level ofoff-target effects. Performing gene editing ex vivo allows one tocharacterize the edited cell population prior to implantation. Thepresent disclosure includes sequencing the entire genome of the editedcells to ensure that the off-target effects, if any, can be in genomiclocations associated with minimal risk to the patient. Furthermore,populations of specific cells, including clonal populations, can beisolated prior to implantation.

Another advantage of ex vivo cell therapy relates to geneticmodification in iPSCs compared to other primary cell sources. iPSCs areprolific, making it easy to obtain the large number of cells that willbe required for a cell-based therapy. Furthermore, iPSCs are an idealcell type for performing clonal isolations. This allows screening forthe correct genomic modification, without risking a decrease inviability. In contrast, other primary cells, such as primary skeletalmuscle cells, are viable for only a few passages and difficult toclonally expand. Thus, manipulation of iPSCs for the treatment ofMyotonic Dystrophy Type 1 can be much easier, and can shorten the amountof time needed to make the desired genetic modification.

Methods can also include an in vivo based therapy. Chromosomal DNA ofthe cells in the patient is edited using the materials and methodsdescribed herein. In some aspects, the target cell in an in vivo basedtherapy is a skeletal muscle cell, a smooth muscle cell, or a cardiacmuscle cell.

Although certain cells present an attractive target for ex vivotreatment and therapy, increased efficacy in delivery may permit directin vivo delivery to such cells. Ideally the targeting and editing wouldbe directed to the relevant cells. Cleavage in other cells can also beprevented by the use of promoters only active in certain cells and ordevelopmental stages. Additional promoters are inducible, and thereforecan be temporally controlled if the nuclease is delivered as a plasmid.The amount of time that delivered RNA and protein remain in the cell canalso be adjusted using treatments or domains added to change thehalf-life. In vivo treatment would eliminate a number of treatmentsteps, but a lower rate of delivery can require higher rates of editing.In vivo treatment can eliminate problems and losses from ex vivotreatment and engraftment.

An advantage of in vivo gene therapy can be the ease of therapeuticproduction and administration. The same therapeutic approach and therapywill have the potential to be used to treat more than one patient, forexample a number of patients who share the same or similar genotype orallele. In contrast, ex vivo cell therapy typically requires using apatient’s own cells, which are isolated, manipulated and returned to thesame patient.

Also provided herein is a cellular method for editing the DMPK gene in acell by genome editing. For example, a cell can be isolated from apatient or animal. Then, the chromosomal DNA of the cell can be editedusing the materials and methods described herein.

The methods provided herein, regardless of whether a cellular or ex vivoor in vivo method, can involve one or a combination of the following: 1)deleting the abnormal repeat expansion within or near the DMPK gene, byinducing two double-stranded DNA breaks at both sides of the expandedregion; 2) deleting the abnormal repeat expansion (in whole or in part)within or near the DMPK gene, by inducing one double-stranded DNA breakproximal to the expanded region; 3) deleting or mutating the DMPK geneby inducing one or more insertions or deletions within or near the DMPKgene or other DNA sequences that encode regulatory elements of the DMPKgene; 4) deleting the mutant DMPK gene and inserting a wild-type DMPKgene, a cDNA or a minigene (comprised of one or more exons and intronsor natural or synthetic introns) into the DMPK gene locus or a safeharbor locus; or 5) targeting a dCas9 fused to chromatin modifyingproteins to DMPK locus to prevent production of transcripts withexpanded repeats.

For example, the dual DSB-induced deletion strategy can involve excisingthe entire abnormal repeat expansion or a portion thereof in the DMPKgene by inducing two or more double stranded breaks at both sides of therepeat region with one or more CRISPR endonucleases and two or moresgRNAs. In certain aspects, a donor DNA containing the correctedsequence can be provided to restore the wild-type sequence. Thisapproach can require development and optimization of sgRNAs and donorDNA molecules for the DMPK gene.

For example, the single DSB-induced deletion strategy can involvedeleting the entire abnormal repeat expansion or a portion thereof inthe DMPK gene by inducing one double stranded break at a site proximalto the repeat region with one or more CRISPR endonucleases and a gRNA(e.g., crRNA + tracrRNA, or sgRNA). In certain aspects, a donor DNAcontaining the corrected sequence can be provided to restore thewild-type sequence. This approach can require development andoptimization of gRNAs and donor DNA molecule for the DMPK gene.

For example, replacing the deleted expanded trinucleotide repeatsequence with a corrected sequence can be achieved by delivering intothe cell one or more CRISPR endonucleases, a pair of gRNAs (e.g.,crRNA + tracrRNA, or sgRNA) targeting upstream or downstream of theexpanded trinucleotide repeat sequence, and a donor DNA that containsthe desired sequence and homology arms to the flanking regions of thetarget locus. This approach can require development and optimization ofsgRNAs for the DMPK gene.

For example, the whole gene correction strategy can involve deletion (inwhole or in part) of the endogenous, mutated DMPK gene and insertion ofa wild-type DMPK gene, a cDNA or a minigene (comprised of one or moreexons and introns or natural or synthetic introns) into the locus of theDMPK gene. It can be achieved by delivering into the cell one or moreCRISPR endonucleases, a pair of gRNAs (e.g., crRNA + tracrRNA, or sgRNA)targeting upstream and downstream of or in the first and last exonand/or intron of the DMPK gene, and a donor DNA that contains thedesired sequence and homology arms to the flanking regions of the targetlocus. The cytogenetic location of the DMPK gene is 19q13.32.Alternatively, the wild-type DMPK gene, a cDNA or a minigene (comprisedof one or more exons and introns or natural or synthetic introns) can beinserted into a safe harbor locus following deletion of the mutant DMPKand expanded repeat sequences. A “safe harbor locus” refers to a regionof the genome where the integrated material can be adequately expressedwithout perturbing endogenous gene structure or function. The safeharbor loci include but are not limited to AAVS1 (PPP1R12C), ALB,Angptl3, ApoC3, ASGR2, CCR5, FIX (F9), Gys2, HGD, Lp(a), Pcsk9,Serpina1, TF, and/or TTR. The target sites within the safe harbor locican be selected from the group consisting of: exon 1-2 of AAVS1(PPP1R12C), exon 1-2 of ALB, exon 1-2 of Angptl3, exon 1-2 of ApoC3,exon 1-2 of ASGR2, exon 1-2 of CCR5, exon 1-2 of FIX (F9), exon 1-2 ofGys2, exon 1-2 of HGD, exon 1-2 of Lp(a), exon 1-2 of Pcsk9, exon 1-2 ofSerpina1, exon 1-2 of TF, and/or exon 1-2 of TTR.

In some aspects, the deletion of the endogenous, mutated DMPK geneincludes deletion of the untranslated regions (UTRs). The donor DNA cancontain corrected UTR sequences.

The whole gene correction strategy utilizes a donor DNA template inHomology-Directed Repair (HDR). HDR may be accomplished by making one ormore single-stranded breaks (SSBs) or double-stranded breaks (DSBs) atspecific sites in the genome by using one or more endonucleases. Thedonor DNA can be single or double stranded DNA. The donor template canhave homologous arms to the 19q13.32 region. The donor template can havehomologous arms to a safe harbor locus. For example, the donor templatecan have homologous arms to an AAVS1 safe harbor locus, such as, intron1 of the PPP1R12C gene.

For example, correcting a point mutation can involve replacing one ormore nucleotide bases, or one or more exons and/or introns within ornear the DMPK gene. Correcting point mutation can involve deleting thesequence containing the mutation by inducing a double stranded break ata site proximal and a site distal to the point mutation with one or moreCRISPR endonucleases and a gRNA (e.g., crRNA + tracrRNA, or sgRNA). Incertain aspects, a donor DNA containing the corrected sequence can beprovided to restore the wild-type sequence. This approach can requiredevelopment and optimization of gRNAs and donor DNA molecule for theDMPK gene.

The advantages for the above strategies are similar, including inprinciple both short and long term beneficial clinical and laboratoryeffects.

In addition to the above genome editing strategies, another strategyinvolves modulating expression, function, or activity of DMPK by editingin the regulatory sequence(s).

In addition to the editing options listed above, Cas9 or similarproteins can be used to target effector domains to the same target sitesthat can be identified for editing, or additional target sites withinrange of the effector domain. A range of chromatin modifying enzymes,methylases or demethylases can be used to alter expression of the targetgene. One possibility is decreasing the expression of the DMPK proteinif the mutation leads to undesirable activity. These types of epigeneticregulation have some advantages, particularly as they are limited inpossible off-target effects.

A number of types of genomic target sites can be present in addition tothe trinucleotide repeat expansion in the non-coding region of the DMPKgene.

The regulation of transcription and translation implicates a number ofdifferent classes of sites that interact with cellular proteins ornucleotides. Often the DNA binding sites of transcription factors orother proteins can be targeted for mutation or deletion to study therole of the site, though they can also be targeted to change geneexpression. Sites can be added through non-homologous end joining NHEJor direct genome editing by homology directed repair (HDR). Increaseduse of genome sequencing, RNA expression and genome-wide studies oftranscription factor binding have increased our ability to identify howthe sites lead to developmental or temporal gene regulation. Thesecontrol systems can be direct or can involve extensive cooperativeregulation that can require the integration of activities from multipleenhancers. Transcription factors typically bind 6-12 bp-long degenerateDNA sequences. The low level of specificity provided by individual sitessuggests that complex interactions and rules are involved in binding andthe functional outcome. Binding sites with less degeneracy can providesimpler means of regulation. Artificial transcription factors can bedesigned to specify longer sequences that have less similar sequences inthe genome and have lower potential for off-target cleavage. Any ofthese types of binding sites can be mutated, deleted or even created toenable changes in gene regulation or expression (Canver, M.C. et al.,Nature (2015)).

Another class of gene regulatory regions having these features ismicroRNA (miRNA) binding sites. miRNAs are non-coding RNAs that play keyroles in posttranscriptional gene regulation. miRNA can regulate theexpression of 30% of all mammalian protein-encoding genes. Specific andpotent gene silencing by double stranded RNA (RNAi) was discovered, plusadditional small non-coding RNA (Canver, M.C. et al., Nature (2015)).The largest class of non-coding RNAs important for gene silencing aremiRNAs. In mammals, miRNAs are first transcribed as a long RNAtranscript, which can be separate transcriptional units, part of proteinintrons, or other transcripts. The long transcripts are called primarymiRNA (pri-miRNA) that include imperfectly base-paired hairpinstructures. These pri-miRNA can be cleaved into one or more shorterprecursor miRNAs (pre-miRNAs) by Microprocessor, a protein complex inthe nucleus, involving Drosha.

Pre-miRNAs are short stem loops ~70 nucleotides in length with a2-nucleotide 3’-overhang that are exported, into the mature 19-25nucleotide miRNA:miRNA* duplexes. The miRNA strand with lower basepairing stability (the guide strand) can be loaded onto the RNA-inducedsilencing complex (RISC). The passenger strand (marked with *), can befunctional, but is usually degraded. The mature miRNA tethers RISC topartly complementary sequence motifs in target mRNAs predominantly foundwithin the 3’ untranslated regions (UTRs) and inducesposttranscriptional gene silencing (Bartel, D.P. Cell 136, 215-233(2009); Saj, A. & Lai, E.C. Curr Opin Genet Dev 21, 504-510 (2011)).

miRNAs can be important in development, differentiation, cell cycle andgrowth control, and in virtually all biological pathways in mammals andother multicellular organisms. miRNAs can also be involved in cell cyclecontrol, apoptosis and stem cell differentiation, hematopoiesis,hypoxia, muscle development, neurogenesis, insulin secretion,cholesterol metabolism, aging, viral replication and immune responses.

A single miRNA can target hundreds of different mRNA transcripts, whilean individual transcript can be targeted by many different miRNAs. Morethan 28645 microRNAs have been annotated in the latest release ofmiRBase (v.21). Some miRNAs can be encoded by multiple loci, some ofwhich can be expressed from tandemly co-transcribed clusters. Thefeatures allow for complex regulatory networks with multiple pathwaysand feedback controls. miRNAs can be integral parts of these feedbackand regulatory circuits and can help regulate gene expression by keepingprotein production within limits (Herranz, H. & Cohen, S.M. Genes Dev24, 1339-1344 (2010); Posadas, D.M. & Carthew, R.W. Curr Opin Genet Dev27, 1-6 (2014)).

miRNA can also be important in a large number of human diseases that areassociated with abnormal miRNA expression. This association underscoresthe importance of the miRNA regulatory pathway. Recent miRNA deletionstudies have linked miRNA with regulation of the immune responses(Stern-Ginossar, N. et al., Science 317, 376-381 (2007)).

miRNA also have a strong link to cancer and can play a role in differenttypes of cancer. miRNAs have been found to be downregulated in a numberof tumors. miRNA can be important in the regulation of keycancer-related pathways, such as cell cycle control and the DNA damageresponse, and can therefore be used in diagnosis and can be targetedclinically. MicroRNAs can delicately regulate the balance ofangiogenesis, such that experiments depleting all microRNAs suppresstumor angiogenesis (Chen, S. et al., Genes Dev 28, 1054-1067 (2014)).

As has been shown for protein coding genes, miRNA genes can also besubject to epigenetic changes occurring with cancer. Many miRNA loci canbe associated with CpG islands increasing their opportunity forregulation by DNA methylation (Weber, B., Stresemann, C., Brueckner, B.& Lyko, F. Cell Cycle 6, 1001-1005 (2007)). The majority of studies haveused treatment with chromatin remodeling drugs to reveal epigeneticallysilenced miRNAs.

In addition to their role in RNA silencing, miRNA can also activatetranslation (Posadas, D.M. & Carthew, R.W. Curr Opin Genet Dev 27, 1-6(2014)). Knocking out these sites may lead to decreased expression ofthe targeted gene, while introducing these sites may increaseexpression.

Individual miRNA can be knocked out most effectively by mutating theseed sequence (bases 2-8 of the microRNA), which can be important forbinding specificity. Cleavage in this region, followed by mis-repair byNHEJ can effectively abolish miRNA function by blocking binding totarget sites. miRNA could also be inhibited by specific targeting of thespecial loop region adjacent to the palindromic sequence. Catalyticallyinactive Cas9 can also be used to inhibit shRNA expression (Zhao, Y. etal., Sci Rep 4, 3943 (2014)). In addition to targeting the miRNA, thebinding sites can also be targeted and mutated to prevent the silencingby miRNA.

According to the present disclosure, any of the microRNA (miRNA) ortheir binding sites may be incorporated into the compositions of thedisclosure.

The compositions may have a region such as, but not limited to, a regioncomprising the sequence of any of the microRNAs listed in SEQ ID NOs:632-4715, the reverse complement of the microRNAs listed in SEQ ID NOs:632-4715, or the microRNA anti-seed region of any of the microRNAslisted in SEQ ID NOs: 632-4715.

The compositions of the present disclosure may comprise one or moremicroRNA target sequences, microRNA sequences, or microRNA seeds. Suchsequences may correspond to any known microRNA such as those taught inU.S. Publication US2005/0261218 and U.S. Publication US2005/0059005. Asa non-limiting example, known microRNAs, their sequences and theirbinding site sequences in the human genome are listed below in SEQ IDNOs: 632-4715.

A microRNA sequence comprises a “seed” region, i.e., a sequence in theregion of positions 2-8 of the mature microRNA, which sequence hasperfect Watson-Crick complementarity to the miRNA target sequence. AmicroRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.In some examples, a microRNA seed may comprise 7 nucleotides (e.g.,nucleotides 2-8 of the mature microRNA), wherein the seed-complementarysite in the corresponding miRNA target is flanked by an adenine (A)opposed to microRNA position 1. In some examples, a microRNA seed maycomprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA),wherein the seed-complementary site in the corresponding miRNA target isflanked by an adenine (A) opposed to microRNA position 1. See forexample, Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP,Bartel DP; Mol Cell. 2007 Jul 6;27(1):91-105. The bases of the microRNAseed have complete complementarity with the target sequence.

Identification of microRNA, microRNA target regions, and theirexpression patterns and role in biology have been reported (Bonauer etal., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr OpinHematol 2011 18:171-176; Contreras and Rao Leukemia 2012 26:404-413(2011 Dec 20. doi: 10.1038/leu.2011.356); Bartel Cell 2009 136:215-233;Landgraf et al, Cell, 2007 129:1401-1414; Gentner and Naldini, TissueAntigens. 2012 80:393-403 and all references therein).

For example, if the composition is not intended to be delivered to theliver but ends up there, then miR-122, a microRNA abundant in liver, caninhibit the expression of the sequence delivered if one or multipletarget sites of miR-122 are engineered into the polynucleotide encodingthat target sequence. Introduction of one or multiple binding sites fordifferent microRNA can be engineered to further decrease the longevity,stability, and protein translation hence providing an additional layerof tenability.

As used herein, the term “microRNA site” refers to a microRNA targetsite or a microRNA recognition site, or any nucleotide sequence to whicha microRNA binds or associates. It should be understood that “binding”may follow traditional Watson-Crick hybridization rules or may reflectany stable association of the microRNA with the target sequence at oradjacent to the microRNA site.

Conversely, for the purposes of the compositions of the presentdisclosure, microRNA binding sites can be engineered out of (i.e.removed from) sequences in which they naturally occur in order toincrease protein expression in specific tissues. For example, miR-206binding sites may be removed to improve protein expression in thecardiac and skeletal muscle.

Specifically, microRNAs are known to be differentially expressed inimmune cells (also called hematopoietic cells), such as antigenpresenting cells (APCs) (e.g. dendritic cells and macrophages),macrophages, monocytes, B lymphocytes, T lymphocytes, granulocytes,natural killer cells, etc. Immune cell specific microRNAs are involvedin immunogenicity, autoimmunity, the immune -response to infection,inflammation, as well as unwanted immune response after gene therapy andtissue/organ transplantation. Immune cells specific microRNAs alsoregulate many aspects of development, proliferation, differentiation andapoptosis of hematopoietic cells (immune cells). For example, miR-142and miR-146 are exclusively expressed in the immune cells, particularlyabundant in myeloid dendritic cells. Introducing the miR-142 bindingsite into the 3’-UTR of a polypeptide of the present disclosure canselectively suppress the gene expression in the antigen presenting cellsthrough miR-142 mediated mRNA degradation, limiting antigen presentationin professional APCs (e.g. dendritic cells) and thereby preventingantigen-mediated immune response after gene delivery (see, Annoni A etal., blood, 2009, 114, 5152-5161).

In one example, microRNAs binding sites that are known to be expressedin immune cells, in particular, the antigen presenting cells, can beengineered into the polynucleotides to suppress the expression of thepolynucleotide in APCs through microRNA mediated RNA degradation,subduing the antigen-mediated immune response, while the expression ofthe polynucleotide is maintained in non-immune cells where the immunecell specific microRNAs are not expressed.

Many microRNA expression studies have been conducted, and are describedin the art, to profile the differential expression of microRNAs invarious cancer cells /tissues and other diseases. Some microRNAs areabnormally over-expressed in certain cancer cells and others areunder-expressed. For example, microRNAs are differentially expressed incancer cells (WO2008/154098, US2013/0059015, US2013/0042333,WO2011/157294); cancer stem cells (US2012/0053224); pancreatic cancersand diseases (US2009/0131348, US2011/0171646, US2010/0286232,US8389210); asthma and inflammation (US8415096); prostate cancer(US2013/0053264); hepatocellular carcinoma (WO2012/151212,US2012/0329672, WO2008/054828, US8252538); lung cancer cells(WO2011/076143, WO2013/033640, WO2009/070653, US2010/0323357); cutaneousT-cell lymphoma (WO2013/011378); colorectal cancer cells(WO2011/0281756, WO2011/076142); cancer positive lymph nodes(WO2009/100430, US2009/0263803); nasopharyngeal carcinoma (EP2112235);chronic obstructive pulmonary disease (US2012/0264626, US2013/0053263);thyroid cancer (WO2013/066678); ovarian cancer cells (US2012/0309645,WO2011/095623); breast cancer cells (WO2008/154098, WO2007/081740,US2012/0214699), leukemia and lymphoma (WO2008/073915, US2009/0092974,US2012/0316081, US2012/0283310, WO2010/018563).

Non-limiting examples of microRNA sequences and the targeted tissuesand/or cells are disclosed in SEQ ID NOs: 632-4715.

Genome Engineering Strategies

The methods of the present disclosure can involve editing one or both ofthe mutant alleles. Gene editing to modify or correct the DMPK gene hasthe advantage of restoration of correct expression levels or eliminationof aberrant gene products and temporal control.

A step of the ex vivo methods of the present disclosure can compriseediting/correcting a skeletal muscle cell, a smooth muscle cell, or acardiac muscle cell isolated from a Myotonic Dystrophy Type 1 patientusing genome engineering. Alternatively, a step of the ex vivo methodsof the present disclosure can comprise editing/correcting the cells ofthe central nervous system, patient specific iPSCs, or mesenchymal stemcells. Likewise, a step of the in vivo methods of the present disclosureinvolves editing/correcting the cells in a Myotonic Dystrophy Type 1patient using genome engineering. Similarly, a step in the cellularmethods of the present disclosure can comprise editing/correcting theDMPK gene in a human cell by genome engineering.

Myotonic Dystrophy Type 1 patients exhibit expanded trinucleotiderepeats in the DMPK gene. Therefore, different patients may use similarediting strategies. Any CRISPR endonuclease may be used in the methodsof the present disclosure, each CRISPR endonuclease having its ownassociated PAM, which may or may not be disease specific.

In one aspect, the trinucleotide repeat expansion may be excised byinducing two double stranded breaks (DSBs) upstream and downstream ofthe repeat region. Pairs of gRNAs have been used for this type ofdeletions. The CRISPR endonucleases, configured with the two gRNAs,induce two DSBs at the desired locations. After cleavage, the two ends,regardless of whether blunt or with overhangs, can be joined by NHEJ,leading to the deletion of the intervening segment. In certain aspects,the wild-type sequence can be restored by inserting a correct repeatsequence via HDR.

In another aspect, the trinucleotide repeat expansion may be deletedafter inducing a single DSB near or within the repeat region. SingleDSB-induced repeat loss has been reported in several studies includingTALEN-cleaved short CAG/CTG repeats engineered in yeast, ZFN-cleaved CAGrepeats engineered in human cells, and CRISPR/Cas9-cleaved CTG/CAGrepeats engineered in human cells (Richard et al., PLoS ONE (2014),9(4): e95611; Mittelman et al., Proc Natl Acad Sci USA (2009),106(24):9607-12; van Agtmaal et al., Mol Ther (2016),http://dx.doi.org/10.1016/j.ymthe.2016.10.014). A DSB near the repeatregion destabilizes the repeat tracts, triggering a contraction (e.g.partial deletion) or, in some cases, a complete deletion of the repeats.Additionally, work by Mittelman et al. suggested that single DSB-inducedrepeat loss is more effective in longer repeats. In certain aspects, thewild-type sequence can be restored by inserting a correct repeatsequence via HDR.

Alternatively, expression of the mutant DMPK gene may be disrupted oreliminated by introducing random insertions or deletions (indels) thatarise due to the imprecise NHEJ repair pathway. The target region may bethe coding sequence of the DMPK gene (i.e., exons). Inserting ordeleting nucleotides into the coding sequence of a gene may cause a“frame shift” where the normal 3-letter codon pattern is disturbed. Inthis way, gene expression and therefore mutant protein production can bereduced or eliminated. This approach may also be used to target anytranscriptional start site, intron, intron:exon junction, or regulatoryDNA element of the DMPK gene where sequence alteration may interferewith the expression of the DMPK gene. This approach can requiredevelopment and optimization of sgRNAs for the DMPK gene.

As a further alternative, the entire mutant gene can be deleted and awild-type gene, a cDNA or a minigene (comprised of one or more exons andintrons or natural or synthetic introns) can be knocked into the genelocus or a heterologous location in the genome such as a safe harborlocus. Pairs of nucleases can be used to delete mutated gene regions,and a donor is provided to restore function. In this case two gRNAs andone donor sequence would be supplied. A full length cDNA can be knockedinto any “safe harbor”, but must use a supplied or an endogenouspromoter. If this construct is knocked into the DMPK gene locus, it willhave physiological control, similar to the normal gene.

Some genome engineering strategies involve replacing the repeatexpansion, or inserting a wild-type DMPK gene or cDNA or a minigene(comprised of one or more exons and introns or natural or syntheticintrons) into the locus of the corresponding gene or a safe harbor locusby homology directed repair (HDR), which is also known as homologousrecombination (HR). Homology directed repair can be one strategy fortreating patients that have expanded trinucleotides in the DMPK gene.These strategies can restore the DMPK gene and reverse, treat, and/ormitigate the diseased state.

Homology directed repair is a cellular mechanism for repairingdouble-stranded breaks (DSBs). The most common form is homologousrecombination. There are additional pathways for HDR, includingsingle-strand annealing and alternative-HDR. Genome engineering toolsallow researchers to manipulate the cellular homologous recombinationpathways to create site-specific modifications to the genome. It hasbeen found that cells can repair a double-stranded break using asynthetic donor molecule provided in trans. Therefore, by introducing adouble-stranded break near a specific mutation and providing a suitabledonor, targeted changes can be made in the genome. Specific cleavageincreases the rate of HDR more than 1,000 fold above the rate of 1 in10⁶ cells receiving a homologous donor alone. The rate of homologydirected repair (HDR) at a particular nucleotide is a function of thedistance to the cut site, so choosing overlapping or nearest targetsites is important. Gene editing offers the advantage over geneaddition, as correcting in situ leaves the rest of the genomeunperturbed.

Supplied donors for editing by HDR vary markedly but can contain theintended sequence with small or large flanking homology arms to allowannealing to the genomic DNA. The homology regions flanking theintroduced genetic changes can be 30 bp or smaller, or as large as amulti-kilobase cassette that can contain promoters, cDNAs, etc. Bothsingle-stranded and double-stranded oligonucleotide donors have beenused. These oligonucleotides range in size from less than 100 nt to overmany kb, though longer ssDNA can also be generated and used.Double-stranded donors can be used, including PCR amplicons, plasmids,and mini-circles. In general, it has been found that an AAV vector canbe a very effective means of delivery of a donor template, though thepackaging limits for individual donors is <5 kb. Active transcription ofthe donor increased HDR three-fold, indicating the inclusion of promotermay increase conversion. Conversely, CpG methylation of the donordecreased gene expression and HDR.

In addition to wild-type endonucleases, such as Cas9, nickase variantsexist that have one or the other nuclease domain inactivated resultingin cutting of only one DNA strand. HDR can be directed from individualCas nickases or using pairs of nickases that flank the target area.Donors can be single-stranded, nicked, or dsDNA.

The donor DNA can be supplied with the nuclease or independently by avariety of different methods, for example by transfection,nano-particle, micro-injection, or viral transduction. A range oftethering options has been proposed to increase the availability of thedonors for HDR. Examples include attaching the donor to the nuclease,attaching to DNA binding proteins that bind nearby, or attaching toproteins that are involved in DNA end binding or repair.

The repair pathway choice can be guided by a number of cultureconditions, such as those that influence cell cycling, or by targetingof DNA repair and associated proteins. For example, to increase HDR, keyNHEJ molecules can be suppressed, such as KU70, KU80 or DNA ligase IV.

Without a donor present, the ends from a DNA break or ends fromdifferent breaks can be joined using the several non-homologous repairpathways in which the DNA ends are joined with little or no base-pairingat the junction. In addition to canonical NHEJ, there are similar repairmechanisms, such as alt-NHEJ. If there are two breaks, the interveningsegment can be deleted or inverted. NHEJ repair pathways can lead toinsertions, deletions or mutations at the joints.

NHEJ was used to insert a 15-kb inducible gene expression cassette intoa defined locus in human cell lines after nuclease cleavage. (See e.g.,Maresca, M., Lin, V.G., Guo, N. & Yang, Y., Genome Res 23, 539-546(2013); Cristea et al. Biotechnology and Bioengineering, 110 (3):871-80(2013); Suzuki et al. Nature, 540, 144-149 (2016)).

In addition to genome editing by NHEJ or HDR, site-specific geneinsertions have been conducted that use both the NHEJ pathway and HR. Acombination approach may be applicable in certain settings, possiblyincluding intron/exon borders. NHEJ may prove effective for ligation inthe intron, while the error-free HDR may be better suited in the codingregion.

The mutation of the DMPK gene that causes Myotonic Dystrophy Type 1 is atrinucleotide repeat expansion of the three letter string of nucleotidesCTG. In healthy individuals, there are few repeats of thistrinucleotide, typically about or fewer than 34. In people with thediseases phenotype, the repeat can occur in the order of thousands. Oneor more trinucleotide repeats may be deleted or corrected in order torestore the gene to a wild-type or similar number of trinucleotiderepeats. In some aspects, all of the trinucleotide repeats may bedeleted. Alternatively, the mutant gene may be knocked out to eliminatetoxic gene products. As a further alternative, following deletion of themutant DMPK allele, a DMPK gene or cDNA can be inserted to the locus ofthe corresponding gene to replace the mutant gene or knocked-in to asafe harbor site, such as AAVS1. In some examples, the methods canprovide one gRNA or a pair of gRNAs that can be used to facilitateincorporation of a new sequence from a polynucleotide donor template toknock-in a part of or the entire DMPK gene or cDNA.

Some genome engineering strategies involve repeat deletion. Targeteddeletion of trinucleotide repeats is an attractive strategy for treatinga large subset of patients with a single therapeutic cocktail. Deletionscan either be single trinucleotide repeat deletions ormulti-trinucleotide repeat deletions. While multi-repeat deletions,including complete deletion of the expanded trinucleotide repeat, canreach a larger number of patients, for larger deletions the efficiencyof deletion greatly decreases with increased size. In some aspects, thedeletions range from 40 to 20,000 base pairs (bp) in size. For example,deletions may range from 40-100; 100-300; 300-500; 500-1,000;1,000-2,000; 2,000-3,000; 3,000-5,000; 5,000-10,000, or 10,000-20,000base pairs in size.

The methods can provide gRNA pairs that make a deletion by cutting thegene twice, one gRNA cutting at the 5’ end of the trinucleotide repeatsand the other gRNA cutting at the 3’ end of the trinucleotide repeats.The cutting can be accomplished by a pair of DNA endonucleases that eachmakes a DSB in the genome, or by multiple nickases that together make aDSB in the genome. The deletion can be followed by insertion of acorrected sequence from a polynucleotide donor template to replace therepeat expansion.

Alternatively, the methods can provide one gRNA to make onedouble-strand cut around the trinucleotide repeats. The double-strandcut can be made by a single DNA endonuclease or multiple nickases thattogether make a DSB in the genome. The deletion can be followed byinsertion of a corrected sequence from a polynucleotide donor templateto replace the repeat expansion.

Illustrative modifications within the DMPK gene include deletions withinor near (proximal) to the trinucleotide repeats referred to above, suchas within the region of less than 3 kb, less than 2 kb, less than 1 kb,less than 0.5 kb upstream or downstream of the trinucleotide repeats.Given the relatively wide variations of trinucleotide repeats in theDMPK gene, it will be appreciated that numerous variations of thedeletions referenced above (including without limitation larger as wellas smaller deletions), would be expected to result in restoration of thewild-type or similar levels of trinucleotide repeats in the DMPK geneexpression.

Such variants can include deletions that are larger in the 5’ and/or 3’direction than the specific repeat expansion in question, or smaller ineither direction. Accordingly, by “near” or “proximal” with respect tospecific repeat expansion, it is intended that the SSB or DSB locusassociated with a desired deletion boundary (also referred to herein asan endpoint) can be within a region that is less than about 3 kb fromthe reference locus noted. The SSB or DSB locus can be more proximal andwithin 2 kb, within 1 kb, within 0.5 kb, or within 0.1 kb. In the caseof small deletion, the desired endpoint can be at or “adjacent to” thereference locus, by which it is intended that the endpoint can be within100 bp, within 50 bp, within 25 bp, or less than about 10 bp to 5 bpfrom the reference locus.

In order to ensure that the pre-mRNA is properly processed followingdeletion, the surrounding splicing signals can be deleted. Splicingdonor and acceptors are generally within 100 base pairs of theneighboring intron. Therefore, in some examples, methods can provide allgRNAs that cut approximately +/- 100-3100 bp with respect to eachexon/intron junction of interest.

For any of the genome editing strategies, gene editing can be confirmedby sequencing or PCR analysis.

Target Sequence Selection

Shifts in the location of the 5’ boundary and/or the 3’ boundaryrelative to particular reference loci can be used to facilitate orenhance particular applications of gene editing, which depend in part onthe endonuclease system selected for the editing, as further describedand illustrated herein.

In a first non-limiting example of such target sequence selection, manyendonuclease systems have rules or criteria that can guide the initialselection of potential target sites for cleavage, such as therequirement of a PAM sequence motif in a particular position adjacent tothe DNA cleavage sites in the case of CRISPR Type II or Type Vendonucleases.

In another non-limiting example of target sequence selection oroptimization, the frequency of off-target activity for a particularcombination of target sequence and gene editing endonuclease (i.e. thefrequency of DSBs occurring at sites other than the selected targetsequence) can be assessed relative to the frequency of on-targetactivity. In some cases, cells that have been correctly edited at thedesired locus can have a selective advantage relative to other cells.Illustrative, but non-limiting, examples of a selective advantageinclude the acquisition of attributes such as enhanced rates ofreplication, persistence, resistance to certain conditions, enhancedrates of successful engraftment or persistence in vivo followingintroduction into a patient, and other attributes associated with themaintenance or increased numbers or viability of such cells. In othercases, cells that have been correctly edited at the desired locus can bepositively selected for by one or more screening methods used toidentify, sort or otherwise select for cells that have been correctlyedited. Both selective advantage and directed selection methods can takeadvantage of the phenotype associated with the correction. In somecases, cells can be edited two or more times in order to create a secondmodification that creates a new phenotype that is used to select orpurify the intended population of cells. Such a second modificationcould be created by adding a second gRNA enabling expression of aselectable or screenable marker. In some cases, cells can be correctlyedited at the desired locus using a DNA fragment that contains the cDNAand also a selectable marker.

Whether any selective advantage is applicable or any directed selectionis to be applied in a particular case, target sequence selection canalso be guided by consideration of off-target frequencies in order toenhance the effectiveness of the application and/or reduce the potentialfor undesired alterations at sites other than the desired target. Asdescribed further and illustrated herein and in the art, the occurrenceof off-target activity can be influenced by a number of factorsincluding similarities and dissimilarities between the target site andvarious off-target sites, as well as the particular endonuclease used.Bioinformatics tools are available that assist in the prediction ofoff-target activity, and frequently such tools can also be used toidentify the most likely sites of off-target activity, which can then beassessed in experimental settings to evaluate relative frequencies ofoff-target to on-target activity, thereby allowing the selection ofsequences that have higher relative on-target activities. Illustrativeexamples of such techniques are provided herein, and others are known inthe art.

Another aspect of target sequence selection relates to homologousrecombination events. Sequences sharing regions of homology can serve asfocal points for homologous recombination events that result in deletionof intervening sequences. Such recombination events occur during thenormal course of replication of chromosomes and other DNA sequences, andalso at other times when DNA sequences are being synthesized, such as inthe case of repairs of double-strand breaks (DSBs), which occur on aregular basis during the normal cell replication cycle but can also beenhanced by the occurrence of various events (such as UV light and otherinducers of DNA breakage) or the presence of certain agents (such asvarious chemical inducers). Many such inducers cause DSBs to occurindiscriminately in the genome, and DSBs can be regularly induced andrepaired in normal cells. During repair, the original sequence can bereconstructed with complete fidelity, however, in some cases, smallinsertions or deletions (referred to as “indels”) are introduced at theDSB site.

DSBs can also be specifically induced at particular locations, as in thecase of the endonucleases systems described herein, which can be used tocause directed or preferential gene modification events at selectedchromosomal locations. The tendency for homologous sequences to besubject to recombination in the context of DNA repair (as well asreplication) can be taken advantage of in a number of circumstances, andis the basis for one application of gene editing systems, such asCRISPR, in which homology directed repair is used to insert a sequenceof interest, provided through use of a “donor” polynucleotide, into adesired chromosomal location.

Regions of homology between particular sequences, which can be smallregions of “microhomology” that can comprise as few as ten base pairs orless, can also be used to bring about desired deletions. For example, asingle DSB can be introduced at a site that exhibits microhomology witha nearby sequence. During the normal course of repair of such DSB, aresult that occurs with high frequency is the deletion of theintervening sequence as a result of recombination being facilitated bythe DSB and concomitant cellular repair process.

In some circumstances, however, selecting target sequences withinregions of homology can also give rise to much larger deletions,including gene fusions (when the deletions are in coding regions), whichmay or may not be desired given the particular circumstances.

The examples provided herein further illustrate the selection of varioustarget regions for the creation of DSBs designed to induce deletion orreplacements that result in restoration of wild-type or similar levelsof trinucleotide repeats in the DMPK gene, as well as the selection ofspecific target sequences within such regions that are designed tominimize off-target events relative to on-target events.

Human Cells

For ameliorating Myotonic Dystrophy Type 1 or any disorder associatedwith DMPK, as described and illustrated herein, the principal targetsfor gene editing are human cells. For example, in the ex vivo methods,the human cells can be somatic cells, which after being modified usingthe techniques as described, can give rise to differentiated cells,e.g., myocytes or progenitor cells. For example, in the in vivo methods,the human cells may be myocytes, neural cells or cells from otheraffected organs.

By performing gene editing in autologous cells that are derived from andtherefore already completely matched with the patient in need, it ispossible to generate cells that can be safely re-introduced into thepatient, and effectively give rise to a population of cells that will beeffective in ameliorating one or more clinical conditions associatedwith the patient’s disease.

Stem cells are capable of both proliferation and giving rise to moreprogenitor cells, these in turn having the ability to generate a largenumber of mother cells that can in turn give rise to differentiated ordifferentiable daughter cells. The daughter cells themselves can beinduced to proliferate and produce progeny that subsequentlydifferentiate into one or more mature cell types, while also retainingone or more cells with parental developmental potential. The term “stemcell” refers then, to a cell with the capacity or potential, underparticular circumstances, to differentiate to a more specialized ordifferentiated phenotype, and which retains the capacity, under certaincircumstances, to proliferate without substantially differentiating. Inone aspect, the term progenitor or stem cell refers to a generalizedmother cell whose descendants (progeny) specialize, often in differentdirections, by differentiation, e.g., by acquiring completely individualcharacters, as occurs in progressive diversification of embryonic cellsand tissues. Cellular differentiation is a complex process typicallyoccurring through many cell divisions. A differentiated cell may derivefrom a multipotent cell that itself is derived from a multipotent cell,and so on. While each of these multipotent cells may be considered stemcells, the range of cell types that each can give rise to may varyconsiderably. Some differentiated cells also have the capacity to giverise to cells of greater developmental potential. Such capacity may benatural or may be induced artificially upon treatment with variousfactors. In many biological instances, stem cells can also be“multipotent” because they can produce progeny of more than one distinctcell type, but this is not required for “stem-ness.”

Self-renewal can be another important aspect of the stem cell. Intheory, self-renewal can occur by either of two major mechanisms. Stemcells can divide asymmetrically, with one daughter retaining the stemstate and the other daughter expressing some distinct other specificfunction and phenotype. Alternatively, some of the stem cells in apopulation can divide symmetrically into two stems, thus maintainingsome stem cells in the population as a whole, while other cells in thepopulation give rise to differentiated progeny only. Generally,“progenitor cells” have a cellular phenotype that is more primitive(i.e., is at an earlier step along a developmental pathway orprogression than is a fully differentiated cell). Often, progenitorcells also have significant or very high-proliferative potential.Progenitor cells can give rise to multiple distinct differentiated celltypes or to a single differentiated cell type, depending on thedevelopmental pathway and on the environment in which the cells developand differentiate.

In the context of cell ontogeny, the adjective “differentiated,” or“differentiating” is a relative term. A “differentiated cell” is a cellthat has progressed further down the developmental pathway than the cellto which it is being compared. Thus, stem cells can differentiate intolineage-restricted precursor cells (such as a myocyte progenitor cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as a myocyte precursor), and then to anend-stage differentiated cell, such as a myocyte, which plays acharacteristic role in a certain tissue type, and may or may not retainthe capacity to proliferate further.

Induced Pluripotent Stem Cells

The genetically engineered human cells described herein can be inducedpluripotent stem cells (iPSCs). An advantage of using iPSCs is that thecells can be derived from the same subject to which the progenitor cellsare to be administered. That is, a somatic cell can be obtained from asubject, reprogrammed to an induced pluripotent stem cell, and thenredifferentiated into a progenitor cell to be administered to thesubject (e.g., autologous cells). Because the progenitors areessentially derived from an autologous source, the risk of engraftmentrejection or allergic response can be reduced compared to the use ofcells from another subject or group of subjects. In addition, the use ofiPSCs negates the need for cells obtained from an embryonic source.Thus, in one aspect, the stem cells used in the disclosed methods arenot embryonic stem cells.

Although differentiation is generally irreversible under physiologicalcontexts, several methods have been recently developed to reprogramsomatic cells to iPSCs. Exemplary methods are known to those of skill inthe art and are described briefly herein below.

The term “reprogramming” refers to a process that alters or reverses thedifferentiation state of a differentiated cell (e.g., a somatic cell).Stated another way, reprogramming refers to a process of driving thedifferentiation of a cell backwards to a more undifferentiated or moreprimitive type of cell. It should be noted that placing many primarycells in culture can lead to some loss of fully differentiatedcharacteristics. Thus, simply culturing such cells included in the termdifferentiated cells does not render these cells nondifferentiated cells(e.g., undifferentiated cells) or pluripotent cells. The transition of adifferentiated cell to pluripotency requires a reprogramming stimulusbeyond the stimuli that lead to partial loss of differentiated characterin culture. Reprogrammed cells also have the characteristic of thecapacity of extended passaging without loss of growth potential,relative to primary cell parents, which generally have capacity for onlya limited number of divisions in culture.

The cell to be reprogrammed can be either partially or terminallydifferentiated prior to reprogramming. Reprogramming can encompasscomplete reversion of the differentiation state of a differentiated cell(e.g., a somatic cell) to a pluripotent state or a multipotent state.Reprogramming can encompass complete or partial reversion of thedifferentiation state of a differentiated cell (e.g., a somatic cell) toan undifferentiated cell (e.g., an embryonic-like cell). Reprogrammingcan result in expression of particular genes by the cells, theexpression of which further contributes to reprogramming. In certainexamples described herein, reprogramming of a differentiated cell (e.g.,a somatic cell) can cause the differentiated cell to assume anundifferentiated state (e.g., is an undifferentiated cell). Theresulting cells are referred to as “reprogrammed cells,” or “inducedpluripotent stem cells (iPSCs or iPS cells).”

Reprogramming can involve alteration, e.g., reversal, of at least someof the heritable patterns of nucleic acid modification (e.g.,methylation), chromatin condensation, epigenetic changes, genomicimprinting, etc., that occur during cellular differentiation.Reprogramming is distinct from simply maintaining the existingundifferentiated state of a cell that is already pluripotent ormaintaining the existing less than fully differentiated state of a cellthat is already a multipotent cell (e.g., a myogenic stem cell).Reprogramming is also distinct from promoting the self-renewal orproliferation of cells that are already pluripotent or multipotent,although the compositions and methods described herein can also be ofuse for such purposes, in some examples.

Many methods are known in the art that can be used to generatepluripotent stem cells from somatic cells. Any such method thatreprograms a somatic cell to the pluripotent phenotype would beappropriate for use in the methods described herein.

Reprogramming methodologies for generating pluripotent cells usingdefined combinations of transcription factors have been described. Mousesomatic cells can be converted to ES cell-like cells with expandeddevelopmental potential by the direct transduction of Oct4, Sox2, Klf4,and c-Myc; see, e.g., Takahashi and Yamanaka, Cell 126(4): 663-76(2006). iPSCs resemble ES cells, as they restore thepluripotency-associated transcriptional circuitry and much of theepigenetic landscape. In addition, mouse iPSCs satisfy all the standardassays for pluripotency: specifically, in vitro differentiation intocell types of the three germ layers, teratoma formation, contribution tochimeras, germline transmission [see, e.g., Maherali and Hochedlinger,Cell Stem Cell. 3(6):595-605 (2008)], and tetraploid complementation.

Human iPSCs can be obtained using similar transduction methods, and thetranscription factor trio, OCT4, SOX2, and NANOG, has been establishedas the core set of transcription factors that govern pluripotency; see,e.g., Budniatzky and Gepstein, Stem Cells Transl Med. 3(4):448-57(2014); Barrett et al., Stem Cells Trans Med 3:1-6 sctm.2014-0121(2014); Focosi et al., Blood Cancer Journal 4: e211 (2014); andreferences cited therein. The production of iPSCs can be achieved by theintroduction of nucleic acid sequences encoding stem cell-associatedgenes into an adult, somatic cell, historically using viral vectors.

iPSCs can be generated or derived from terminally differentiated somaticcells, as well as from adult stem cells, or somatic stem cells. That is,a non-pluripotent progenitor cell can be rendered pluripotent ormultipotent by reprogramming. In such instances, it may not be necessaryto include as many reprogramming factors as required to reprogram aterminally differentiated cell. Further, reprogramming can be induced bythe non-viral introduction of reprogramming factors, e.g., byintroducing the proteins themselves, or by introducing nucleic acidsthat encode the reprogramming factors, or by introducing messenger RNAsthat upon translation produce the reprogramming factors (see e.g.,Warren et al., Cell Stem Cell, 7(5):618-30 (2010). Reprogramming can beachieved by introducing a combination of nucleic acids encoding stemcell-associated genes, including, for example, Oct-4 (also known asOct-¾ or Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klfl, Klf2,Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN28.Reprogramming using the methods and compositions described herein canfurther comprise introducing one or more of Oct-¾, a member of the Soxfamily, a member of the Klf family, and a member of the Myc family to asomatic cell. The methods and compositions described herein can furthercomprise introducing one or more of each of Oct-4, Sox2, Nanog, c-MYCand Klf4 for reprogramming. As noted above, the exact method used forreprogramming is not necessarily critical to the methods andcompositions described herein. However, where cells differentiated fromthe reprogrammed cells are to be used in, e.g., human therapy, in oneaspect the reprogramming is not effected by a method that alters thegenome. Thus, in such examples, reprogramming can be achieved, e.g.,without the use of viral or plasmid vectors.

The efficiency of reprogramming (i.e., the number of reprogrammed cells)derived from a population of starting cells can be enhanced by theaddition of various agents, e.g., small molecules, as shown by Shi etal., Cell-Stem Cell 2:525-528 (2008); Huangfu et al., NatureBiotechnology 26(7):795-797 (2008) and Marson et al., Cell-Stem Cell 3:132-135 (2008). Thus, an agent or combination of agents that enhance theefficiency or rate of induced pluripotent stem cell production can beused in the production of patient-specific or disease-specific iPSCs.Some non-limiting examples of agents that enhance reprogrammingefficiency include soluble Wnt, Wnt conditioned media, BIX-01294 (a G9ahistone methyltransferase), PD0325901 (a MEK inhibitor), DNAmethyltransferase inhibitors, histone deacetylase (HDAC) inhibitors,valproic acid, 5’-azacytidine, dexamethasone, suberoylanilide,hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others.

Other non-limiting examples of reprogramming enhancing agents include:Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) andother hydroxamic acids), BML-210, Depudecin (e.g., (-)-Depudecin), HCToxin, Nullscript(4-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide),Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VP A)and other short chain fatty acids), Scriptaid, Suramin Sodium,Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate,pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin,Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994(e.g., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA(m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin,A-161906, proxamide, oxamflatin, 3-Cl-UCHA (e.g.,6-(3-chlorophenylureido) caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50. Other reprogrammingenhancing agents include, for example, dominant negative forms of theHDACs (e.g., catalytically inactive forms), siRNA inhibitors of theHDACs, and antibodies that specifically bind to the HDACs. Suchinhibitors are available, e.g., from BIOMOL International, Fukasawa,Merck Biosciences, Novartis, Gloucester Pharmaceuticals, TitanPharmaceuticals, MethylGene, and Sigma Aldrich.

To confirm the induction of pluripotent stem cells for use with themethods described herein, isolated clones can be tested for theexpression of a stem cell marker. Such expression in a cell derived froma somatic cell identifies the cells as induced pluripotent stem cells.Stem cell markers can be selected from the non-limiting group includingSSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto,Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl. In one case, for example, acell that expresses Oct4 or Nanog is identified as pluripotent. Methodsfor detecting the expression of such markers can include, for example,RT-PCR and immunological methods that detect the presence of the encodedpolypeptides, such as Western blots or flow cytometric analyses.Detection can involve not only RT-PCR, but can also include detection ofprotein markers. Intracellular markers may be best identified viaRT-PCR, or protein detection methods such as immunocytochemistry, whilecell surface markers are readily identified, e.g., byimmunocytochemistry.

The pluripotent stem cell character of isolated cells can be confirmedby tests evaluating the ability of the iPSCs to differentiate into cellsof each of the three germ layers. As one example, teratoma formation innude mice can be used to evaluate the pluripotent character of theisolated clones. The cells can be introduced into nude mice andhistology and/or immunohistochemistry can be performed on a tumorarising from the cells. The growth of a tumor comprising cells from allthree germ layers, for example, further indicates that the cells arepluripotent stem cells.

Muscle Cells

In some aspects, the genetically engineered human cells described hereinare myocytes, i.e. cells of the muscle. The muscle is composed of threetypes of tissue, namely, cardiac, smooth, and skeletal. Cardiac musclecells are located in the walls of the heart and are under involuntarycontrol. Smooth muscle fibers are located in the walls of hollowvisceral organs, except the heart, and are also under involuntarycontrol. On the other hand, skeletal muscle fibers are under voluntarycontrol and are located in muscles which are attached to the skeleton.

Cells of the Central Nervous System

In some aspects, the genetically engineered human cells described hereinare cells of the central nervous system. Neurons, which processinformation, and glial cells (or neuroglia), which provide mechanicaland metabolic support to the nervous system and modulate informationprocessed by neurons, are the two main classes of cells of the centralnervous system. Non-limiting examples of neurons include sensory neurons(also referred to as afferent neurons) that transfer information fromthe external environment to the central nervous system, motor neurons(also referred to as efferent neurons) that transfer information fromthe central nervous system to the external environment, and interneurons(also referred to as association neurons) that process information inthe central nervous system and transfers the information from one neuronto the other within the central nervous system. Non-limiting examples ofglial cells include astrocytes, oligodendrocytes, microglia and Schwanncells. CNS progenitor cells can be neural progenitor cells or glialprogenitor cells.

The typical neuron transmits electrical signals from one cell toanother. Neurons contain a cell body, dendrite, axon hillock, axon,nerve endings, neuronal synapses, and neuromuscular junctions. Neuronsmay be named according to shape or the nature of the dendritic tree.

Neuroglia differ from neurons in several general ways in that they: donot form synapses, have essentially only one type of process, retain theability to divide, and are less electrically excitable than neurons.Neuroglia are classified based on size and shape of their nucleus anddistinguished from neurons, at the light microscopic level. Neurogliaare divided into two major categories based on size, the macroglia andthe microglia. The macroglia are of ectodermal origin and consist ofastrocytes, oligodendrocytes and ependymal cells. Microglia cells areprobably of mesodermal origin.

Creating Patient Specific iPSCs

One step of the ex vivo methods of the present disclosure can involvecreating a patient specific iPS cell, patient specific iPS cells, or apatient specific iPS cell line. There are many established methods inthe art for creating patient specific iPS cells, as described inTakahashi and Yamanaka 2006; Takahashi, Tanabe et al. 2007. For example,the creating step can comprise: a) isolating a somatic cell, such as askin cell or fibroblast, from the patient; and b) introducing a set ofpluripotency-associated genes into the somatic cell in order to inducethe cell to become a pluripotent stem cell. The set ofpluripotency-associated genes can be one or more of the genes selectedfrom the group consisting of OCT4, SOX1, SOX2, SOX3, SOX15, SOX18,NANOG, KLF1, KLF2, KLF4, KLF5, c-MYC, n-MYC, REM2, TERT and LIN28.

Performing a Biopsy or Aspirate of the Patient’s Tissue

A biopsy or aspirate is a sample of tissue or fluid taken from the body.There are many different kinds of biopsies or aspirates. Nearly all ofthem involve using a sharp tool to remove a small amount of tissue. Ifthe biopsy will be on the skin or other sensitive area, numbing medicinecan be applied first. A biopsy or aspirate may be performed according toany of the known methods in the art. For example, in a bone marrowaspirate, a large needle is used to enter the pelvis bone to collectbone marrow. For example, in a needle biopsy of the muscle, a needle isinserted into the muscle. A small piece of tissue is retained in theneedle when it is removed. For example, in an open biopsy of the muscle,a small cut is made in the skin and into the muscle, following which themuscle tissue is removed.

Isolating a Myocyte

Myocytes from the skeletal muscle may be isolated according to anymethod known in the art. For example, the isolated muscle is placed intoa dish with 0.2% collagenase I solution and incubated for 2.5 hours. Themuscle is dissected using Pasteur pipettes pre-flushed with HS solution.Using forceps, tendons and their attached myofibers are separated. Theclumps of myofibers are triturated to separate out individual singlemyofibers. The myofibers are triturated further and eventually purified.The final myofiber suspension obtained is added to 35-mm culture dishescoated with isotonic Pure Col collagen and the dishes are incubated fora minimum of 20-30 minutes to allow formation of PureCol collagen matrixand the attachment of the myofibers to the matrix. The culture issupplied with myofiber culture medium, cells are cultured andcharacterized for expression of skeletal muscle markers such as Myf5,MyoD, myogenin, MYH2 (myosin heavy chain-2 protein) and alpha-tubulinprotein (Keire et al., Methods in Molecular Biology, 2013; 946:431-468.)

Myocytes from the cardiac muscle, i.e. cardiomyocytes may be isolatedaccording to any method known in the art. For example, human myocytesare isolated during routine cannulation procedures in patients whoundergo open-heart surgery. Cardiomyocytes from the atria are isolatedfrom the tip of the right atrial appendage. The tissue is excised andtransferred into a petri dish with sterile Ca²⁺- free transport solutioncontaining 2,3-butanedione monoxime and is dissected by chopping it intosmall chunks. The resulting mixture is stirred with a magnetic stirringbar and the supernatant obtained is strained through a 200 µm nylonmesh. The tissue chunks are subjected to a first enzymatic digestion ofcollagenase and protease and a second enzymatic digestion of collagenaseI. The resulting supernatant is strained through a 200 µm mesh again andthe cells are further disassociated by mechanical trituration via a 20ml serological pipette with dispenser. The supernatant obtained isstrained through a 200 µm nylon mesh again and centrifuged. The pelletobtained is resuspended in storage solution and 10 mM CaCl₂ solution.The cardiomyocytes are analyzed further for characterization of markerssuch as TNNT2, and NKX2-5 and cultured (Voight et al., Journal ofVisualized Experiments, 2013;77:e50235).

Myocytes from the smooth muscle may be isolated according to any methodknown in the art. For example, a biopsy of the artery is performed andthe artery, for example the aorta, is further dissected into smallpieces. Explants with vessel lumens facing down are then placed in100-mm culture plates. The explants are supplied with Dulbecco’smodified eagle medium (DMEM) supplemented with 10% fetal bovine serum(FBS) along with antibiotics and antimycotics. Culture plates areincubated at 37° C. and cultured for 4 weeks, at which point the cellsare confluent. Cells are grown in DMEM without serum for 24 hours atthis stage. Cells are supplemented back with FBS and characterized forexpression of markers such as alpha-smooth muscle actin, and smoothmuscle myosin (Leik et al., Hypertension, 2004;43:837-840).

Isolating a Mesenchymal Stem Cell

Mesenchymal stem cells can be isolated according to any method known inthe art, such as from a patient’s bone marrow or peripheral blood. Forexample, marrow aspirate can be collected into a syringe with heparin.Cells can be washed and centrifuged on a Percoll. The cells can becultured in Dulbecco’s modified Eagle’s medium (DMEM) (low glucose)containing 10% fetal bovine serum (FBS) (Pittinger MF, Mackay AM, BeckSC et al., Science 1999; 284:143-147).

Genetically Modified Cells

The term “genetically modified cell” refers to a cell that comprises atleast one genetic modification introduced by genome editing (e.g., usingthe CRISPR/Cas9 or CRISPR/Cpfl system). In some ex vivo examples herein,the genetically modified cell can be genetically modified myogenicprogenitor cell. In some in vivo examples herein, the geneticallymodified cell can be a genetically modified skeletal muscle cell. Agenetically modified cell comprising an exogenous genome-targetingnucleic acid and/or an exogenous nucleic acid encoding agenome-targeting nucleic acid is contemplated herein.

The term “control treated population” describes a population of cellsthat has been treated with identical media, viral induction, nucleicacid sequences, temperature, confluency, flask size, pH, etc., with theexception of the addition of the genome editing components. Any methodknown in the art can be used to measure restoration of DMPK gene orprotein expression or activity, for example Western Blot analysis of theDMPK protein or real time PCR for quantifying DMPK mRNA.

The term “isolated cell” refers to a cell that has been removed from anorganism in which it was originally found, or a descendant of such acell. Optionally, the cell can be cultured in vitro, e.g., under definedconditions or in the presence of other cells. Optionally, the cell canbe later introduced into a second organism or re-introduced into theorganism from which it (or the cell from which it is descended) wasisolated.

The term “isolated population” with respect to an isolated population ofcells refers to a population of cells that has been removed andseparated from a mixed or heterogeneous population of cells. In somecases, the isolated population can be a substantially pure population ofcells, as compared to the heterogeneous population from which the cellswere isolated or enriched. In some cases, the isolated population can bean isolated population of human progenitor cells, e.g., a substantiallypure population of human progenitor cells, as compared to aheterogeneous population of cells comprising human progenitor cells andcells from which the human progenitor cells were derived.

The term “substantially enhanced,” with respect to a particular cellpopulation, refers to a population of cells in which the occurrence of aparticular type of cell is increased relative to pre-existing orreference levels, by at least 2-fold, at least 3-, at least 4-, at least5-, at least 6-, at least 7-, at least 8-, at least 9, at least 10-, atleast 20-, at least 50-, at least 100-, at least 400-, at least 1000-,at least 5000-, at least 20000-, at least 100000- or more folddepending, e.g., on the desired levels of such cells for amelioratingMyotonic Dystrophy Type 1.

The term “substantially enriched” with respect to a particular cellpopulation, refers to a population of cells that is at least about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or morewith respect to the cells making up a total cell population.

The term “substantially pure” with respect to a particular cellpopulation, refers to a population of cells that is at least about 75%,at least about 85%, at least about 90%, or at least about 95% pure, withrespect to the cells making up a total cell population. That is, theterms “substantially pure” or “essentially purified,” with regard to apopulation of progenitor cells, refers to a population of cells thatcontain fewer than about 20%, about 15%, about 10%, about 9%, about 8%,about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, orless than 1%, of cells that are not progenitor cells as defined by theterms herein.

Differentiation of Genome-Edited iPSCs Into Muscle Cells or Cells of theCentral Nervous System (CNS)

Another step of the ex vivo methods of the present disclosure cancomprise differentiating the genome-edited iPSCs into skeletal musclecells, smooth muscle cells, cardiac muscle cells, or Pax7+ muscleprogenitor cells. The differentiating step may be performed according toany method known in the art. For example, genome-edited iPSCs aredifferentiated into myocytes using various treatments, including MyoD,ETV2, FLI1, and ERG1. For example, genome-edited iPSCs aredifferentiated into cardiomyocytes using various treatments, includingascorbic acid, BMP4, GSK-inhibitor, BMP-inhibitor, Wnt/β-cateninsignalling inhibitor. For example, genome-edited iPSCs aredifferentiated into vascular smooth muscle cells using varioustreatments, including development of embryoid bodies from genome-editediPSCs, followed by differentiation of embryoid bodies into myogeniccells.

Another step of the ex vivo methods of the present disclosure cancomprise differentiating the genome-edited iPSC into cells of thecentral nervous system (CNS) (e.g., neurons or glial cells). Thedifferentiating step may be performed according to any method known inthe art.

Differentiation of Genome-Edited Mesenchymal Stem Cells Into MuscleCells or Cells of the Central Nervous System (CNS)

Another step of the ex vivo methods of the present disclosure cancomprise differentiating the genome-edited mesenchymal stem cells intoskeletal muscle cells, smooth muscle cells, or cardiac muscle cells, orPax7+ muscle progenitor cells. The differentiating step may be performedaccording to any method known in the art. For example, hMSCs are treatedwith various factors and hormones, including culturing the hMSCs withculture medium consisting of DMEM/Ham’s F-12 with 10% FBS and 1%L-glutamine, culturing the hMSCs with differentiation medium containingDMEM/Ham’s F12, 2% donor horse serum, 1% L-glutamine, 1 ng/mL bFGF, and0.4 µg/mL dexamethasone.

Another step of the ex vivo methods of the present disclosure cancomprise differentiating the genome-edited mesenchymal stem into cellsof the central nervous system (e.g., neurons or glial cells). Thedifferentiating step may be performed according to any method known inthe art.

Implanting Cells Into Patients

Another step of the ex vivo methods of the present disclosure cancomprise implanting the skeletal muscle cells, smooth muscle cells,cardiac muscle cells, or Pax7+ muscle progenitor cells into patients.This implanting step may be accomplished using any method ofimplantation known in the art. For example, the genetically modifiedcells may be injected directly in the patient’s blood or injected intothe desired muscle, or otherwise administered to the patient.

Another step of the ex vivo methods of the present disclosure involvesimplanting the cells of the central nervous system (e.g., neurons or aglial cells) into patients. This implanting step may be accomplishedusing any method of implantation known in the art. For example, thegenetically modified neurons may be administered to the patient viaintraparenchymal, vascular (e.g., intravenous, intra-arterial), orventricular (e.g., intracerebroventricular, intracisternal, intrathecal)routes or other routes such as intracranial or intraperitonealinjection.

III. Formulations and Delivery Pharmaceutically Acceptable Carriers

The ex vivo methods of administering progenitor cells to a subjectcontemplated herein involve the use of therapeutic compositionscomprising progenitor cells.

Therapeutic compositions can contain a physiologically tolerable carriertogether with the cell composition, and optionally at least oneadditional bioactive agent as described herein, dissolved or dispersedtherein as an active ingredient. In some cases, the therapeuticcomposition is not substantially immunogenic when administered to amammal or human patient for therapeutic purposes, unless so desired.

In general, the progenitor cells described herein can be administered asa suspension with a pharmaceutically acceptable carrier. One of skill inthe art will recognize that a pharmaceutically acceptable carrier to beused in a cell composition will not include buffers, compounds,cryopreservation agents, preservatives, or other agents in amounts thatsubstantially interfere with the viability of the cells to be deliveredto the subject. A formulation comprising cells can include e.g., osmoticbuffers that permit cell membrane integrity to be maintained, andoptionally, nutrients to maintain cell viability or enhance engraftmentupon administration. Such formulations and suspensions are known tothose of skill in the art and/or can be adapted for use with theprogenitor cells, as described herein, using routine experimentation.

A cell composition can also be emulsified or presented as a liposomecomposition, provided that the emulsification procedure does notadversely affect cell viability. The cells and any other activeingredient can be mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient, and in amountssuitable for use in the therapeutic methods described herein.

Additional agents included in a cell composition can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids, such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases, such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH value, physiological salineor both, such as phosphate-buffered saline. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes. Liquid compositions can also contain liquid phases in additionto and to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions. The amount of an active compound used in the cellcompositions that is effective in the treatment of a particular disorderor condition can depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques.

Guide RNA Formulation

Guide RNAs of the present disclosure can be formulated withpharmaceutically acceptable excipients such as carriers, solvents,stabilizers, adjuvants, diluents, etc., depending upon the particularmode of administration and dosage form. Guide RNA compositions can beformulated to achieve a physiologically compatible pH, and range from apH of about 3 to a pH of about 11, about pH 3 to about pH 7, dependingon the formulation and route of administration. In some cases, the pHcan be adjusted to a range from about pH 5.0 to about pH 8. In somecases, the compositions can comprise a therapeutically effective amountof at least one compound as described herein, together with one or morepharmaceutically acceptable excipients. Optionally, the compositions cancomprise a combination of the compounds described herein, or can includea second active ingredient useful in the treatment or prevention ofbacterial growth (for example and without limitation, anti-bacterial oranti-microbial agents), or can include a combination of reagents of thepresent disclosure.

Suitable excipients include, for example, carrier molecules that includelarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, and inactive virus particles. Otherexemplary excipients can include antioxidants (for example and withoutlimitation, ascorbic acid), chelating agents (for example and withoutlimitation, EDTA), carbohydrates (for example and without limitation,dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose),stearic acid, liquids (for example and without limitation, oils, water,saline, glycerol and ethanol), wetting or emulsifying agents, pHbuffering substances, and the like.

Delivery

Guide RNA polynucleotides (RNA or DNA) and/or endonucleasepolynucleotide(s) (RNA or DNA) can be delivered by viral or non-viraldelivery vehicles known in the art. Alternatively, endonucleasepolypeptide(s) can be delivered by viral or non-viral delivery vehiclesknown in the art, such as electroporation or lipid nanoparticles. Infurther alternative aspects, the DNA endonuclease can be delivered asone or more polypeptides, either alone or pre-complexed with one or moreguide RNAs, or one or more crRNA together with a tracrRNA.

Polynucleotides can be delivered by non-viral delivery vehiclesincluding, but not limited to, nanoparticles, liposomes,ribonucleoproteins, positively charged peptides, small moleculeRNA-conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes.Some exemplary non-viral delivery vehicles are described in Peer andLieberman, Gene Therapy, 18: 1127-1133 (2011) (which focuses onnon-viral delivery vehicles for siRNA that are also useful for deliveryof other polynucleotides).

For polynucleotides of the disclosure, the formulation may be selectedfrom any of those taught, for example, in International ApplicationPCT/US2012/069610.

Polynucleotides, such as guide RNA, sgRNA, and mRNA encoding anendonuclease, may be delivered to a cell or a patient by a lipidnanoparticle (LNP).

A LNP refers to any particle having a diameter of less than 1000 nm, 500nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.Alternatively, a nanoparticle may range in size from 1-1000 nm, 1-500nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

LNPs may be made from cationic, anionic, or neutral lipids. Neutrallipids, such as the fusogenic phospholipid DOPE or the membranecomponent cholesterol, may be included in LNPs as ‘helper lipids’ toenhance transfection activity and nanoparticle stability. Limitations ofcationic lipids include low efficacy owing to poor stability and rapidclearance, as well as the generation of inflammatory oranti-inflammatory responses.

LNPs may also be comprised of hydrophobic lipids, hydrophilic lipids, orboth hydrophobic and hydrophilic lipids.

Any lipid or combination of lipids that are known in the art can be usedto produce a LNP. Examples of lipids used to produce LNPs are: DOTMA,DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol,GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG).Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2-DMA (KC2),DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are:DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are:PEG-DMG, PEG-CerC14, and PEG-CerC20.

The lipids can be combined in any number of molar ratios to produce aLNP. In addition, the polynucleotide(s) can be combined with lipid(s) ina wide range of molar ratios to produce a LNP.

As stated previously, the site-directed polypeptide and genome-targetingnucleic acid can each be administered separately to a cell or a patient.On the other hand, the site-directed polypeptide can be pre-complexedwith one or more guide RNAs, or one or more crRNA together with atracrRNA. The pre-complexed material can then be administered to a cellor a patient. Such pre-complexed material is known as aribonucleoprotein particle (RNP).

RNA is capable of forming specific interactions with RNA or DNA. Whilethis property is exploited in many biological processes, it also comeswith the risk of promiscuous interactions in a nucleic acid-richcellular environment. One solution to this problem is the formation ofribonucleoprotein particles (RNPs), in which the RNA is pre-complexedwith an endonuclease. Another benefit of the RNP is protection of theRNA from degradation.

The endonuclease in the RNP can be modified or unmodified. Likewise, thegRNA, crRNA, tracrRNA, or sgRNA can be modified or unmodified. Numerousmodifications are known in the art and can be used.

The endonuclease and sgRNA can be generally combined in a 1:1 molarratio. Alternatively, the endonuclease, crRNA and tracrRNA can begenerally combined in a 1:1:1 molar ratio. However, a wide range ofmolar ratios can be used to produce a RNP.

AAV (Adeno Associated Virus)

A recombinant adeno-associated virus (AAV) vector can be used fordelivery. Techniques to produce rAAV particles, in which an AAV genometo be packaged that includes the polynucleotide to be delivered, rep andcap genes, and helper virus functions are provided to a cell, arestandard in the art. Production of rAAV typically requires that thefollowing components are present within a single cell (denoted herein asa packaging cell): a rAAV genome, AAV rep and cap genes separate from(i.e., not in) the rAAV genome, and helper virus functions. The AAV repand cap genes may be from any AAV serotype for which recombinant viruscan be derived, and may be from a different AAV serotype than the rAAVgenome ITRs, including, but not limited to, AAV serotypes describedherein. Production of pseudotyped rAAV is disclosed in, for example,international patent application publication number WO 01/83692.

AAV Serotypes

AAV particles packaging polynucleotides encoding compositions of thedisclosure, e.g., endonucleases, donor sequences, or RNA guidemolecules, of the present disclosure may comprise or be derived from anynatural or recombinant AAV serotype. According to the presentdisclosure, the AAV particles may utilize or be based on a serotypeselected from any of the following serotypes, and variants thereofincluding but not limited to AAV1, AAV10, AAV106.1hu.37, AAV11,AAV114.3/hu.40, AAV12, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.1hu.43,AAV128.3/hu.44, AAV130.4/hu.48, AAV145.⅟hu.53, AAV145.5/hu.54,AAV145.6/hu.55, AAV16.12/hu.11, AAV16.3, AAV16.8/hu.10, AAV161.10/hu.60,AAV161.6/hu.61, AAV1-7/rh.48, AAV1-8/rh.49, AAV2, AAV2.5T,AAV2-15/rh.62, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6,AAV223.7, AAV2-3/rh.61, AAV24.1, AAV2-4/rh.50, AAV2-5/rh.51, AAV27.3,AAV29.3/bb.1, AAV29.5/bb.2, AAV2G9, AAV-2-pre-miRNA-101, AAV3,AAV3.1hu.6, AAV3.1hu.9, AAV3-11/rh.53, AAV3-3, AAV33.12/hu.17,AAV33.4/hu.15, AAV33.8/hu.16, AAV3-9/rh.52, AAV3a, AAV3b, AAV4,AAV4-19/rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13,AAV42-15, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a,AAV42-5b, AAV42-6b, AAV42-8, AAV42-aa, AAV43-1, AAV43-12, AAV43-20,AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2,AAV44.5, AAV46.2/hu.28, AAV46.6/hu.29, AAV4-8/r11.64, AAV4-8/rh.64,AAV4-9/rh.54, AAV5, AAV52.1hu.20, AAV52/hu.19, AAV5-22/rh.58,AAV5-3/rh.57, AAV54.1hu.21, AAV54.2/hu.22, AAV54.4R/hu.27,AAV54.5/hu.23, AAV54.7/hu.24, AAV58.2/hu.25, AAV6, AAV6.1, AAV6.1.2,AAV6.2, AAV7, AAV7.2, AAV7.3/hu.7, AAV8, AAV-8b, AAV-8h, AAV9, AAV9.11,AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84,AAV9.9, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAV-b, AAVC1, AAVC2, AAVC5,AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1,AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAV-DJ, AAV-DJ8, AAVF3, AAVF5,AAV-h, AAVH-1hu.1, AAVH2, AAVH-5/hu.3, AAVH6, AAVhE1.1, AAVhER1.14,AAVhEr1.16, AAVhEr1.18, AAVhER1.23, AAVhEr1.35, AAVhEr1.36, AAVhEr1.5,AAVhEr1.7, AAVhEr1.8, AAVhEr2.16, AAVhEr2.29, AAVhEr2.30, AAVhEr2.31,AAVhEr2.36, AAVhEr2.4, AAVhEr3.1, AAVhu.1, AAVhu.10, AAVhu.11, AAVhu.11,AAVhu.12, AAVhu.13, AAVhu.14/9, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18,AAVhu.19, AAVhu.2, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24,AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.3, AAVhu.31,AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.4, AAVhu.40,AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2,AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1,AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.5, AAVhu.51, AAVhu.52, AAVhu.53,AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.6, AAVhu.60,AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.7, AAVhu.8,AAVhu.9, AAVhu.t 19, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39,AAVLG-9/hu.39, AAV-LK01, AAV-LK02, AAVLK03, AAV-LK03, AAV-LK04,AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11,AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK17, AAV-LK18, AAV-LK19,AAVN721-8/rh.43, AAV-PAEC, AAV-PAEC11, AAV-PAEC12, AAV-PAEC2, AAV-PAEC4,AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAVpi.1, AAVpi.2, AAVpi.3, AAVrh.10,AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19,AAVrh.2, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25,AAVrh.2R, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36,AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.44,AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.48, AAVrh.48.1,AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.50, AAVrh.51, AAVrh.52,AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59,AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64R1, AAVrh.64R2,AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73,AAVrh.74, AAVrh.8, AAVrh.8R, AAVrh8R, AAVrh8R A586R mutant, AAVrh8RR533A mutant, BAAV, BNP61 AAV, BNP62 AAV, BNP63 AAV, bovine AAV, caprineAAV, Japanese AAV 10, true type AAV (ttAAV), UPENN AAV 10, AAV-LK16,AAAV, AAV Shuffle 100-1, AAV Shuffle 100-2, AAV Shuffle 100-3, AAVShuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAVSM 100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, and/or AAV SM 10-8.

In some aspects, the AAV serotype may be, or have, a mutation in theAAV9 sequence as described by N Pulicherla et al. (Molecular Therapy19(6):1070-1078 (2011)), such as but not limited to, AAV9.9, AAV9.11,AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84.

In some aspects, the AAV serotype may be, or have, a sequence asdescribed in U.S. Pat. No. U.S. 6156303, such as, but not limited to,AAV3B (SEQ ID NO: 1 and 10 of U.S. 6156303), AAV6 (SEQ ID NO: 2, 7 and11 of U.S. 6156303), AAV2 (SEQ ID NO: 3 and 8 of U.S. 6156303), AAV3A(SEQ ID NO: 4 and 9, of U.S. 6156303), or derivatives thereof.

In some aspects, the serotype may be AAVDJ or a variant thereof, such asAAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal of Virology82(12): 5887-5911 (2008)). The amino acid sequence of AAVDJ8 maycomprise two or more mutations in order to remove the heparin bindingdomain (HBD). As a non-limiting example, the AAV-DJ sequence describedas SEQ ID NO: 1 in U.S. Pat. No. 7,588,772, may comprise two mutations:(1) R587Q where arginine (R; Arg) at amino acid 587 is changed toglutamine (Q; Gln) and (2) R590T where arginine (R; Arg) at amino acid590 is changed to threonine (T; Thr). As another non-limiting example,may comprise three mutations: (1) K406R where lysine (K; Lys) at aminoacid 406 is changed to arginine (R; Arg), (2) R587Q where arginine (R;Arg) at amino acid 587 is changed to glutamine (Q; Gln) and (3) R590Twhere arginine (R; Arg) at amino acid 590 is changed to threonine (T;Thr).

In some aspects, the AAV serotype may be, or have, a sequence asdescribed in International Publication No. WO2015121501, such as, butnot limited to, true type AAV (ttAAV) (SEQ ID NO: 2 of WO2015121501),“UPenn AAV10” (SEQ ID NO: 8 of WO2015121501), “Japanese AAV10” (SEQ IDNO: 9 of WO2015121501), or variants thereof.

According to the present disclosure, AAV capsid serotype selection oruse may be from a variety of species. In one example, the AAV may be anavian AAV (AAAV). The AAAV serotype may be, or have, a sequence asdescribed in U.S. Pat. No. U.S. 9238800, such as, but not limited to,AAAV (SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, and 14 of U.S. 9,238,800), orvariants thereof.

In one example, the AAV may be a bovine AAV (BAAV). The BAAV serotypemay be, or have, a sequence as described in U.S. Pat. No. U.S.9,193,769, such as, but not limited to, BAAV (SEQ ID NO: 1 and 6 of U.S.9193769), or variants thereof. The BAAV serotype may be or have asequence as described in U.S. Pat. No. U.S.7427396, such as, but notlimited to, BAAV (SEQ ID NO: 5 and 6 of U.S.7427396), or variantsthereof.

In one example, the AAV may be a caprine AAV. The caprine AAV serotypemay be, or have, a sequence as described in U.S. Pat. No. U.S. 7427396,such as, but not limited to, caprine AAV (SEQ ID NO: 3 of U.S. 7427396),or variants thereof.

In other examples the AAV may be engineered as a hybrid AAV from two ormore parental serotypes. In one example, the AAV may be AAV2G9 whichcomprises sequences from AAV2 and AAV9. The AAV2G9 AAV serotype may be,or have, a sequence as described in U.S. Pat. Publication No.US20160017005.

In one example, the AAV may be a serotype generated by the AAV9 capsidlibrary with mutations in amino acids 390-627 (VP1 numbering) asdescribed by Pulicherla et al. (Molecular Therapy 19(6):1070-1078(2011). The serotype and corresponding nucleotide and amino acidsubstitutions may be, but is not limited to, AAV9.1 (G1594C; D532H),AAV6.2 (T1418A and T1436X; V473D and I479K), AAV9.3 (T1238A; F413Y),AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G,C1760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F411I), AAV9.9 (G1203A,G1785T; W595C), AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T,A1702C, A1769T; T568P, Q590L), AAV9.13 (A1369C, A1720T; N457H, T574S),AAV9.14 (T1340A, T1362C, T1560C, G1713A; L447H), AAV9.16 (A1775T;Q592L), AAV9.24 (T1507C, T1521G; W503R), AAV9.26 (A1337G, A1769C; Y446C,Q590P), AAV9.33 (A1667C; D556A), AAV9.34 (A1534G, C1794T; N512D),AAV9.35 (A1289T, T1450A, C1494T, A1515T, C1794A, G1816A; Q430L, Y484N,N98K, V606I), AAV9.40 (A1694T, E565V), AAV9.41 (A1348T, T1362C; T450S),AAV9.44 (A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T;N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W509R, L517V),9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I),AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50 (A1638T, C1683T, T1805A;Q546H, L602H), AAV9.53 (G1301A, A1405C, C1664T, G1811T; R134Q, S469R,A555V, G604V), AAV9.54 (C1531A, T1609A; L511I, L537M), AAV9.55 (T1605A;F535L), AAV9.58 (C1475T, C1579A; T492I, H527N), AAV.59 (T1336C; Y446H),AAV9.61 (A1493T; N498I), AAV9.64 (C1531A, A1617T; L511I), AAV9.65(C1335T, T1530C, C1568A; A523D), AAV9.68 (C1510A; P504T), AAV9.80(G1441A,;G481R), AAV9.83 (C1402A, A1500T; P468T, E500D), AAV9.87(T1464C, T1468C; S490P), AAV9.90 (A1196T; Y399F), AAV9.91 (T1316G,A1583T, C1782G, T1806C; L439R, K528I), AAV9.93 (A1273G, A1421G, A1638C,C1712T, G1732A, A1744T, A1832T; S425G, Q474R, Q546H, P571L, G578R,T582S, D611V), AAV9.94 (A1675T; M559L) and AAV9.95 (T1605A; F535L).

In one example, the AAV may be a serotype comprising at least one AAVcapsid CD8+ T-cell epitope. As a non-limiting example, the serotype maybe AAV1, AAV2 or AAV8.

In one example, the AAV may be a variant, such as PHP.A or PHP.B asdescribed in Deverman. 2016. Nature Biotechnology. 34(2): 204-209.

In one example, the AAV may be a serotype selected from any of thosefound in SEQ ID NOs: 4734-5302 and Table 2.

In one example, the AAV may be encoded by a sequence, fragment orvariant as disclosed in SEQ ID NOs: 4734-5302 and Table 2.

General principles of rAAV production are reviewed in, for example,Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka,1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Variousapproaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072(1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984);Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J.Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol.,7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat.No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243(PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark etal. (1996) Gene Therapy 3:1124-1132; U.S. Pat. No. 5,786,211; U.S.Patent No. 5,871,982; and U.S. Pat. No. 6,258,595.

AAV vector serotypes can be matched to target cell types. For example,the following exemplary cell types can be transduced by the indicatedAAV serotypes among others.

TABLE 2 Tissue/Cell Types and Serotypes Tissue/Cell Type Serotype LiverAAV3, AAV8, AA5, AAV9 Skeletal muscle AAV1, AAV7, AAV6, AAV8, AAV9Central nervous system AAV5, AAV1, AAV4, AAV9 RPE AAV5, AAV4Photoreceptor cells AAV5 Lung AAV9 Heart AAV8 Pancreas AAV8 Kidney AAV2,AA8 Hematopoietic stem cells AAV6

In addition to adeno-associated viral vectors, other viral vectors canbe used. Such viral vectors include, but are not limited to, lentivirus,alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, EpsteinBarr virus, papovavirus, poxvirus, vaccinia virus, and herpes simplexvirus.

In some aspects, Cas9 mRNA, sgRNA targeting one or two sites in DMPKgene, and donor DNA can each be separately formulated into lipidnanoparticles, or are all co-formulated into one lipid nanoparticle.

In some aspects, Cas9 mRNA can be formulated in a lipid nanoparticle,while sgRNA and donor DNA can be delivered in an AAV vector.

Options are available to deliver the Cas9 nuclease as a DNA plasmid, asmRNA or as a protein. The guide RNA can be expressed from the same DNA,or can also be delivered as an RNA. The RNA can be chemically modifiedto alter or improve its half-life, or decrease the likelihood or degreeof immune response. The endonuclease protein can be complexed with thegRNA prior to delivery. Viral vectors allow efficient delivery; splitversions of Cas9 and smaller orthologs of Cas9 can be packaged in AAV,as can donors for HDR. A range of non-viral delivery methods also existthat can deliver each of these components, or non-viral and viralmethods can be employed in tandem. For example, nano-particles can beused to deliver the protein and guide RNA, while AAV can be used todeliver a donor DNA.

In some aspects of the in vivo based therapy described herein, the viralvector(s) encoding the endonuclease, guide RNA and/or donor DNA may bedelivered to the skeletal muscle, smooth muscle or cardiac muscle bymeans of local, regional, or systemic administration.

IV. Dosing and Administration

The terms “administering,” “introducing” and “transplanting” are usedinterchangeably in the context of the placement of cells, e.g.,progenitor cells, into a subject, by a method or route that results inat least partial localization of the introduced cells at a desired site,such as a site of injury or repair, such that a desired effect(s) isproduced. The cells e.g., progenitor cells, or their differentiatedprogeny can be administered by any appropriate route that results indelivery to a desired location in the subject where at least a portionof the implanted cells or components of the cells remain viable. Theperiod of viability of the cells after administration to a subject canbe as short as a few hours, e.g., twenty-four hours, to a few days, toas long as several years, or even the life time of the patient, i.e.,long-term engraftment. For example, in some aspects described herein, aneffective amount of myogenic progenitor cells is administered via asystemic route of administration, such as an intraperitoneal orintravenous route.

The terms “individual,” “subject,” “host” and “patient” are usedinterchangeably herein and refer to any subject for whom diagnosis,treatment or therapy is desired. In some aspects, the subject is amammal. In some aspects, the subject is a human being.

When provided prophylactically, progenitor cells described herein can beadministered to a subject in advance of any symptom of MyotonicDystrophy Type 1. Accordingly, the prophylactic administration of aprogenitor cell population serves to prevent Myotonic Dystrophy Type 1.

A progenitor cell population being administered according to the methodsdescribed herein can comprise allogeneic progenitor cells obtained fromone or more donors. Such progenitors may be of any cellular or tissueorigin, e.g., liver, muscle, cardiac, etc. “Allogeneic” refers to aprogenitor cell or biological samples comprising progenitor cellsobtained from one or more different donors of the same species, wherethe genes at one or more loci are not identical. For example, a liverprogenitor cell population being administered to a subject can bederived from one more unrelated donor subjects, or from one or morenon-identical siblings. In some cases, syngeneic progenitor cellpopulations can be used, such as those obtained from geneticallyidentical animals, or from identical twins. The progenitor cells can beautologous cells; that is, the progenitor cells are obtained or isolatedfrom a subject and administered to the same subject, i.e., the donor andrecipient are the same.

The term “effective amount” refers to the amount of a population ofprogenitor cells or their progeny needed to prevent or alleviate atleast one or more signs or symptoms of Myotonic Dystrophy Type 1, andrelates to a sufficient amount of a composition to provide the desiredeffect, e.g., to treat a subject having Myotonic Dystrophy Type 1. Theterm “therapeutically effective amount” therefore refers to an amount ofprogenitor cells or a composition comprising progenitor cells that issufficient to promote a particular effect when administered to a typicalsubject, such as one who has or is at risk for Myotonic DystrophyType 1. An effective amount would also include an amount sufficient toprevent or delay the development of a symptom of the disease, alter thecourse of a symptom of the disease (for example but not limited to, slowthe progression of a symptom of the disease), or reverse a symptom ofthe disease. It is understood that for any given case, an appropriate“effective amount” can be determined by one of ordinary skill in the artusing routine experimentation.

For use in the various aspects described herein, an effective amount ofprogenitor cells comprises at least 10² progenitor cells, at least 5 X10² progenitor cells, at least 10³ progenitor cells, at least 5 X 10³progenitor cells, at least 10⁴ progenitor cells, at least 5 X 10⁴progenitor cells, at least 10⁵ progenitor cells, at least 2 X 10⁵progenitor cells, at least 3 X 10⁵ progenitor cells, at least 4 X 10⁵progenitor cells, at least 5 X 10⁵ progenitor cells, at least 6 X 10⁵progenitor cells, at least 7 X 10⁵ progenitor cells, at least 8 X 10⁵progenitor cells, at least 9 X 10⁵ progenitor cells, at least 1 X 10⁶progenitor cells, at least 2 X 10⁶ progenitor cells, at least 3 X 10⁶progenitor cells, at least 4 X 10⁶ progenitor cells, at least 5 X 10⁶progenitor cells, at least 6 X 10⁶ progenitor cells, at least 7 X 10⁶progenitor cells, at least 8 X 10⁶ progenitor cells, at least 9 X 10⁶progenitor cells, or multiples thereof. The progenitor cells can bederived from one or more donors, or can be obtained from an autologoussource. In some examples described herein, the progenitor cells can beexpanded in culture prior to administration to a subject in needthereof.

In some aspects, reduction of the expanded trinucleotide repeats in theDMPK gene in cells of patients having a DMPK related disorder can bebeneficial for ameliorating one or more symptoms of the disease, forincreasing long-term survival, and/or for reducing side effectsassociated with other treatments. Upon administration of such cells tohuman patients, the presence of progenitors that have wild-type orsimilar levels of trinucleotide repeat in the DMPK gene is beneficial.In some cases, effective treatment of a subject gives rise to at leastabout 3%, 5% or 7% transcripts having wild-type or similar levels oftrinucleotide repeat relative to total DMPK transcripts in the treatedsubject. In some examples, transcripts having wild-type or similarlevels of trinucleotide repeat will be at least about 10% of total DMPKtranscripts. In some examples, transcripts having wild-type or similarlevels of trinucleotide repeat will be at least about 20% to 30% oftotal DMPK transcripts. Similarly, the introduction of even relativelylimited subpopulations of cells having wild-type levels of trinucleotiderepeats in the DMPK gene can be beneficial in various patients becausein some situations normalized cells will have a selective advantagerelative to diseased cells. However, even modest levels of progenitorswith transcripts having wild-type or similar levels of trinucleotiderepeat in the DMPK gene can be beneficial for ameliorating one or moreaspects of Myotonic Dystrophy Type 1 in patients. In some examples,about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about70%, about 80%, about 90% or more of the myogenic progenitors inpatients to whom such cells are administered have wild-type or similarlevels of trinucleotide repeat in the DMPK gene.

“Administered” refers to the delivery of a progenitor cell compositioninto a subject by a method or route that results in at least partiallocalization of the cell composition at a desired site. A cellcomposition can be administered by any appropriate route that results ineffective treatment in the subject, i.e. administration results indelivery to a desired location in the subject where at least a portionof the composition delivered, i.e. at least 1 x 10⁴ cells are deliveredto the desired site for a period of time.

In one aspect of the method, the pharmaceutical composition may beadministered via a route such as, but not limited to, enteral (into theintestine), gastroenteral, epidural (into the dura matter), oral (by wayof the mouth), transdermal, peridural, intracerebral (into thecerebrum), intracerebroventricular (into the cerebral ventricles),epicutaneous (application onto the skin), intradermal, (into the skinitself), subcutaneous (under the skin), nasal administration (throughthe nose), intravenous (into a vein), intravenous bolus, intravenousdrip, intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection (into a pathologic cavity)intracavitary (into the base of the penis), intravaginal administration,intrauterine, extra-amniotic administration, transdermal (diffusionthrough the intact skin for systemic distribution), transmucosal(diffusion through a mucous membrane), transvaginal, insufflation(snorting), sublingual, sublabial, enema, eye drops (onto theconjunctiva), in ear drops, auricular (in or by way of the ear), buccal(directed toward the cheek), conjunctival, cutaneous, dental (to a toothor teeth), electro-osmosis, endocervical, endosinusial, endotracheal,extracorporeal, hemodialysis, infiltration, interstitial,intra-abdominal, intraamniotic, intra-articular, intrabiliary,intrabronchial, intrabursal, intracartilaginous (within a cartilage),intracaudal (within the cauda equine), intracisternal (within thecisterna magna cerebellomedularis), intracorneal (within the cornea),dental intracornal, intracoronary (within the coronary arteries),intracorporus cavernosum (within the dilatable spaces of the corporuscavernosa of the penis), intradiscal (within a disc), intraductal(within a duct of a gland), intraduodenal (within the duodenum),intradural (within or beneath the dura), intraepidermal (to theepidermis), intraesophageal (to the esophagus), intragastric (within thestomach), intragingival (within the gingivae), intraileal (within thedistal portion of the small intestine), intralesional (within orintroduced directly to a localized lesion), intraluminal (within a lumenof a tube), intralymphatic (within the lymph), intramedullary (withinthe marrow cavity of a bone), intrameningeal (within the meninges),intramyocardial (within the myocardium), intraocular (within the eye),intraovarian (within the ovary), intrapericardial (within thepericardium), intrapleural (within the pleura), intraprostatic (withinthe prostate gland), intrapulmonary (within the lungs or its bronchi),intrasinal (within the nasal or periorbital sinuses), intraspinal(within the vertebral column), intrasynovial (within the synovial cavityof a joint), intratendinous (within a tendon), intratesticular (withinthe testicle), intrathecal (within the cerebrospinal fluid at any levelof the cerebrospinal axis), intrathoracic (within the thorax),intratubular (within the tubules of an organ), intratumor (within atumor), intratympanic (within the aurus media), intravascular (within avessel or vessels), intraventricular (within a ventricle), iontophoresis(by means of electric current where ions of soluble salts migrate intothe tissues of the body), irrigation (to bathe or flush open wounds orbody cavities), laryngeal (directly upon the larynx), nasogastric(through the nose and into the stomach), occlusive dressing technique(topical route administration, which is then covered by a dressing thatoccludes the area), ophthalmic (to the external eye), oropharyngeal(directly to the mouth and pharynx), parenteral, percutaneous,periarticular, peridural, perineural, periodontal, rectal, respiratory(within the respiratory tract by inhaling orally or nasally for local orsystemic effect), retrobulbar (behind the pons or behind the eyeball),intramyocardial (entering the myocardium), soft tissue, subarachnoid,subconjunctival, submucosal, topical, transplacental (through or acrossthe placenta), transtracheal (through the wall of the trachea),transtympanic (across or through the tympanic cavity), ureteral (to theureter), urethral (to the urethra), vaginal, caudal block, diagnostic,nerve block, biliary perfusion, cardiac perfusion, photopheresis andspinal.

Modes of administration include injection, infusion, instillation,and/or ingestion. “Injection” includes, without limitation, intravenous,intramuscular, intra-arterial, intrathecal, intraventricular,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular,subarachnoid, intraspinal, intracerebro spinal, and intrasternalinjection and infusion. In some examples, the route is intravenous. Forthe delivery of cells, administration by injection or infusion can bemade.

The cells can be administered systemically. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” refer to theadministration of a population of progenitor cells other than directlyinto a target site, tissue, or organ, such that it enters, instead, thesubject’s circulatory system and, thus, is subject to metabolism andother like processes.

The efficacy of a treatment comprising a composition for the treatmentof Myotonic Dystrophy Type 1 can be determined by the skilled clinician.However, a treatment is considered “effective treatment,” if any one orall of the signs or symptoms of, as but one example, levels oftrinucleotide repeat in the DMPK gene are altered in a beneficial manner(e.g., decreased by at least 10%), or other clinically accepted symptomsor markers of disease are improved or ameliorated. Efficacy can also bemeasured by failure of an individual to worsen as assessed byhospitalization or need for medical interventions (e.g., progression ofthe disease is halted or at least slowed). Methods of measuring theseindicators are known to those of skill in the art and/or describedherein. Treatment includes any treatment of a disease in an individualor an animal (some non-limiting examples include a human, or a mammal)and includes: (1) inhibiting the disease, e.g., arresting, or slowingthe progression of symptoms; or (2) relieving the disease, e.g., causingregression of symptoms; and (3) preventing or reducing the likelihood ofthe development of symptoms.

The treatment according to the present disclosure can ameliorate one ormore symptoms associated with Myotonic Dystrophy Type 1 by reducing thenumber of trinucleotide repeat in the DMPK gene in the individual.

V. Features and Properties of the Dystrophia Myotonica-Protein Kinase(DMPK) Gene

DMPK has been associated with diseases and disorders such as, but notlimited to, Atherosclerosis, Azoospermia, Hypertrophic Cardiomyopathy,Celiac Disease, Congenital chromosomal disease, Diabetes Mellitus, Focalglomerulosclerosis, Huntington Disease, Hypogonadism, Muscular Atrophy,Myopathy, Muscular Dystrophy, Myotonia, Myotonic Dystrophy,Neuromuscular Diseases, Optic Atrophy, Paresis, Schizophrenia, Cataract,Spinocerebellar Ataxia, Muscle Weakness, Adrenoleukodystrophy,Centronuclear myopathy, Interstitial fibrosis, myotonic musculardystrophy, Abnormal mental state, X-linked Charcot-Marie-Tooth disease1, Congenital Myotonic Dystrophy, Bilateral cataracts (disorder),Congenital Fiber Type Disproportion, Myotonic Disorders, Multisystemdisorder, 3-Methylglutaconic aciduria type 3, cardiac event, CardiogenicSyncope, Congenital Structural Myopathy, Mental handicap,Adrenomyeloneuropathy, Dystrophia myotonica 2, and IntellectualDisability. Editing the DMPK gene using any of the methods describedherein may be used to treat, prevent and/or mitigate the symptoms of thediseases and disorders described herein.

The activity of DMPK plays an important role in muscle, heart, and braincells, and DMPK is associated with Myotonic Dystrophy Type 1 (DM1),which is also known as muscular dystrophy. DM1 results from expansion ofa CTG trinucleotide repeat in the 3’ untranslated region (UTR) of theDMPK gene. In most people, the number of CTG repeats ranges from 5 to34. Individuals with CTG expansions from 35-49 repeats do not havesymptoms, but their children are at increased risk of developing thisdisorder because the repeats are likely to expand during meiosis.Individuals with DM1 have from 50 to 5,000 CTG repeats in most cells.The repeat counts may be even greater in certain types of cells, such asmuscle cells. The mutated DMPK gene produces an altered mRNA, whichaccumulates in the cell and interferes with the production of many otherproteins.

DM1 is an inherited disease (autosomal dominant) where the myotonicdystrophy protein kinase has been shown to turn off (inhibit) part of amuscle protein called myosin phosphatase. Myosin phosphatase is anenzyme that plays a role in muscle tensing (contraction) and relaxation.DM1 affects between 1 in 100,000 people in populations of Japan to 1 in10,000 people in Iceland. In the United States the incidence of DM1 isestimated to be about 1 in 8,000 people worldwide. Common symptoms ofDM1 include muscle weakness and wasting, prolonged muscle tensing(myotonia), cataracts, and arrhythmias. No specific treatment for themuscle weakness associated with DM1 is currently available. Although,treatment is available for resulting disorders such as diabetesmellitus, cataracts, and hypothyroidism.

In one example, the target tissue for the compositions and methodsdescribed herein is muscle tissue, such as but not limited to skeletalmuscle, smooth muscle, and cardiac muscle. In another example, thetarget tissue for the compositions and methods described herein iscentral nervous system tissue.

In one example, the gene is Dystrophia Myotonica-Protein Kinase (DMPK)which may also be referred to as Myotonic Dystrophy Associated ProteinKinase, Myotonin Protein Kinase A, Thymopoietin Homolog, DM1 ProteinKinase, DM Protein Kinase, EC 2.7.11.1, DM1PK, and MT-PK. DMPK has acytogenetic location of 19q13.32 and the genomic coordinate are onChromosome 19 on the forward strand at position 45,769,717-45,782,552.The nucleotide sequence of DMPK is shown as SEQ ID NO: 5303. AC011530.4is a gene upstream of and overlapping with DMPK on the reverse strand,and DMWD is also a gene upstream of DMPK. SIX5 is the gene downstream ofDMPK on the reverse strand. AC074212.6 and AC074212.5are the geneslocated on the forward strand opposite of DMPK. DMPK has a NCBI gene IDof 1760, Uniprot ID of Q09013 and Ensembl Gene ID of ENSG00000104936.DMPK has 1249 SNPs, 65 introns and 70 exons. The exon identifier fromEnsembl and the start/stop sites of the introns and exons are shown inTable 3.

TABLE 3 Introns and Exons for DMPK Exon No. Exon ID Start/Stop IntronNo. Intron based on Exon ID Start/Stop EX1 ENSE00001475019 45,780,603-45,779,778 INT1 Intron ENSE00001475019 -ENSE00003515823 45,779,777-45,779,523 EX2 ENSE00003515823 45,779,522 -45,779,439 INT2 IntronENSE00003515823 -ENSE00003672131 45,779,438 -45,779,360 EX3ENSE00003672131 45,779,359 -45,779,264 INT3 Intron ENSE00003672131-ENSE00003494201 45,779,263 -45,778,642 EX4 ENSE00003494201 45,778,641-45,778,493 INT4 Intron ENSE00003494201 -ENSE00003534388 45,778,492-45,778,221 EX5 ENSE00003534388 45,778,220 -45,778,127 INT5 IntronENSE00003534388 -ENSE00003484367 45,778,126 -45,777,874 EX6ENSE00003484367 45,777,873 -45,777,667 INT6 Intron ENSE00003484367-ENSE00003609541 45,777,666 -45,777,591 EX7 ENSE00003609541 45,777,590-45,777,327 INT7 Intron ENSE00003609541 -ENSE00003525807 45,777,326-45,775,035 EX8 ENSE00003525807 45,775,034 -45,774,949 INT8 IntronENSE00003525807 -ENSE00003653656 45,774,948 -45,772,753 EX9ENSE00003653656 45,772,752 -45,772,641 INT9 Intron ENSE00003653656-ENSE00003494269 45,772,640 -45,771,929 EX10 ENSE00003494269 45,771,928-45,771,771 INT10 Intron ENSE00003494269 -ENSE00003592282 45,771,770-45,771,666 EX11 ENSE00003592282 45,771,665 -45,771,568 INT11 IntronENSE00003592282 -ENSE00003610901 45,771,567 -45,771,397 EX12ENSE00003610901 45,771,396 -45,771,350 INT12 Intron ENSE00003610901-ENSE00003615528 45,771,349 -45,771,061 EX13 ENSE00003615528 45,771,060-45,770,971 INT13 Intron ENSE00003615528 -ENSE00003216639 45,770,970-45,770,641 EX14 ENSE00003216639 45,770,640 -45,769,725 INT14 IntronENSE00002858732 -ENSE00003554377 45,782,192 -45,779,870 EX15ENSE00002858732 45,782,478 -45,782,193 INT15 Intron ENSE00003554377-ENSE00003515823 45,779,777 -45,779,523 EX16 ENSE00003554377 45,779,869-45,779,778 INT16 Intron ENSE00003615528 -ENSE00003640441 45,770,970-45,770,641 EX17 ENSE00003640441 45,770,640 -45,769,717 INT17 IntronENSE00002742422 -ENSE00003554377 45,782,192 -45,779,870 EX18ENSE00002742422 45,782,479 -45,782,193 INT18 Intron ENSE00003484367-ENSE00001157000 45,777,666 -45,777,591 EX19 ENSE00001157000 45,777,590-45,777,342 INT19 Intron ENSE00001157000 -ENSE00003525807 45,777,341-45,775,035 EX20 ENSE00002811897 45,770,640 -45,769,736 INT20 IntronENSE00003615528 -ENSE00002811897 45,770,970 -45,770,641 EX21ENSE00002970823 45,782,388 -45,782,193 INT21 Intron ENSE00002970823-ENSE00003554377 45,782,192 -45,779,870 EX22 ENSE00003663498 45,771,056-45,770,971 INT22 Intron ENSE00003610901 -ENSE00003663498 45,771,349-45,771,057 EX23 ENSE00002918999 45,770,640 -45,769,847 INT23 IntronENSE00003663498 -ENSE00002918999 45,770,970 -45,770,641 EX24ENSE00003726218 45,770,640 -45,770,250 INT24 Intron ENSE00003663498-ENSE00003726218 45,770,970 -45,770,641 EX25 ENSE00003749267 45,770,204-45,769,847 INT25 Intron ENSE00003726218 -ENSE00003749267 45,770,249-45,770,205 EX26 ENSE00002307404 45,782,489 -45,782,193 INT26 IntronENSE00002307404 -ENSE00003554377 45,782,192 -45,779,870 EX27ENSE00002258162 45,770,640 -45,770,381 INT27 Intron ENSE00003592282-ENSE00002258162 45,771,567 -45,770,641 EX28 ENSE00003080618 45,780,747-45,779,778 INT28 Intron ENSE00003080618 -ENSE00003515823 45,779,777-45,779,523 EX29 ENSE00003577916 45,770,640 -45,769,720 INT29 IntronENSE00003663498 -ENSE00003577916 45,770,970 -45,770,641 EX30ENSE00003058631 45,772,712 -45,772,641 INT30 Intron ENSE00003058631-ENSE00003494269 45,772,640 -45,771,929 EX31 ENSE00003084759 45,771,396-45,770,922 INT31 Intron ENSE00003592282 -ENSE00003084759 45,771,567-45,771,397 EX32 ENSE00003217233 45,777,447 -45,777,327 INT32 IntronENSE00003217233 -ENSE00003525807 45,777,326 -45,775,035 EX33ENSE00003665789 45,771,665 -45,771,568 INT33 Intron ENSE00003653656-ENSE00003665789 45,772,640 -45,771,666 EX34 ENSE00003124522 45,770,640-45,770,510 INT34 Intron ENSE00003665789 -ENSE00003124522 45,771,567-45,770,641 EX35 ENSE00003159664 45,780,541 -45,780,323 INT35 IntronENSE00003159664 -ENSE00003554377 45,780,322 -45,779,870 EX36ENSE00003036149 45,778,641 -45,778,635 INT36 Intron ENSE00003672131-ENSE00003036149 45,779,263 -45,778,642 EX37 ENSE00001112734 45,782,552-45,782,193 INT37 Intron ENSE00001112734 -ENSE00002857960 45,782,192-45,780,382 EX38 ENSE00002857960 45,780,381 -45,780,323 INT38 IntronENSE00002857960 -ENSE00003495366 45,780,322 -45,779,870 EX39ENSE00003495366 45,779,869 -45,779,778 INT39 Intron ENSE00003495366-ENSE00003582547 45,779,777 -45,779,523 EX40 ENSE00003582547 45,779,522-45,779,439 INT40 Intron ENSE00003582547 -ENSE00003664445 45,779,438-45,779,360 EX41 ENSE00003664445 45,779,359 -45,779,264 INT41 IntronENSE00003664445 -ENSE00003642705 45,779,263 -45,778,642 EX42ENSE00003642705 45,778,641 -45,778,493 INT42 Intron ENSE00003642705-ENSE00003671885 45,778,492 -45,778,221 EX43 ENSE00003671885 45,778,220-45,778,127 INT43 Intron ENSE00003671885 -ENSE00003591961 45,778,126-45,777,874 EX44 ENSE00003591961 45,777,873 -45,777,667 INT44 IntronENSE00003591961 -ENSE00003636370 45,777,666 -45,777,591 EX45ENSE00003636370 45,777,590 -45,777,327 INT45 Intron ENSE00003636370-ENSE00003633227 45,777,326 -45,775,035 EX46 ENSE00003633227 45,775,034-45,774,949 INT46 Intron ENSE00003633227 -ENSE00003508373 45,774,948-45,772,753 EX47 ENSE00003508373 45,772,752 -45,772,641 INT47 IntronENSE00003508373 -ENSE00003531496 45,772,640 -45,771,929 EX48ENSE00003531496 45,771,928 -45,771,771 INT48 Intron ENSE00003531496-ENSE00003475037 45,771,770 -45,771,666 EX49 ENSE00003475037 45,771,665-45,771,568 INT49 Intron ENSE00003475037 -ENSE00003670737 45,771,567-45,771,397 EX50 ENSE00003670737 45,771,396 -45,771,350 INT50 IntronENSE00003670737 -ENSE00003577920 45,771,349 -45,771,061 EX51ENSE00003577920 45,771,060 -45,770,971 INT51 Intron ENSE00003577920-ENSE00003672714 45,770,970 -45,770,641 EX52 ENSE00003672714 45,770,640-45,769,717 INT52 Intron ENSE00003131421 -ENSE00002998597 45,776,658-45,775,035 EX53 ENSE00003131421 45,777,037 -45,776,659 INT53 IntronENSE00003182972 -ENSE00003642705 45,779,133 -45,778,642 EX54ENSE00002998597 45,775,034 -45,774,948 INT54 Intron ENSE00003671885-ENSE00003099899 45,778,126 -45,777,874 EX55 ENSE00003182972 45,779,168-45,779,134 INT55 Intron ENSE00003206314 -ENSE00003664445 45,779,438-45,779,360 EX56 ENSE00003099899 45,777,873 -45,777,770 INT56 IntronENSE00003670737 -ENSE00003629157 45,771,349 -45,771,057 EX57ENSE00003206314 45,779,619 -45,779,439 INT57 Intron ENSE00003629157-ENSE00003624574 45,770,970 -45,770,641 EX58 ENSE00003629157 45,771,056-45,770,971 INT58 Intron ENSE00003014430 -ENSE00003582547 45,779,777-45,779,523 EX59 ENSE00003624574 45,770,640 -45,769,720 INT59 IntronENSE00003582547 -ENSE00003211453 45,779,438 -45,779,360 EX60ENSE00003014430 45,780,506 -45,779,778 INT60 Intron ENSE00002993785-ENSE00003671885 45,778,492 -45,778,221 EX61 ENSE00003211453 45,779,359-45,778,547 INT61 Intron ENSE00003671885 -ENSE00003076450 45,778,126-45,777,874 EX62 ENSE00003074123 45,771,365 -45,769,956 INT62 IntronENSE00003204067 -ENSE00003508373 45,774,948 -45,772,753 EX63ENSE00002993785 45,778,950 -45,778,493 INT63 Intron ENSE00003531496-ENSE00003014995 45,771,770 -45,771,666 EX64 ENSE00003076450 45,777,873-45,777,694 INT64 Intron ENSE00003043777 -ENSE00003151461 45,779,438-45,779,360 EX65 ENSE00003204067 45,775,268 -45,774,949 INT65 IntronENSE00003104165 -ENSE00003196899 45,777,666 -45,777,591 EX66ENSE00003014995 45,771,665 -45,771,533 EX67 ENSE00003043777 45,779,569-45,779,439 EX68 ENSE00003151461 45,779,359 -45,778,903 EX69ENSE00003104165 45,777,954 -45,777,667 EX70 ENSE00003196899 45,777,590-45,777,566

Table 4 provides information on all of the transcripts for the DMPK genebased on the Ensembl database. Provided in Table 4 are the transcript IDfrom Ensembl and corresponding NCBI RefSeq ID for the transcript, thetranslation ID from Ensembl and the corresponding NCBI RefSeq ID for theprotein, the biotype of the transcript sequence as classified by Ensembland the exons and introns in the transcript based on the information inTable 3.

TABLE 4 Transcript Information for DMPK Transcript ID Transcript NCBIRefSeq ID Translation ID Protein NCBI RefSeq ID Sequence Biotype Exon IDfrom Table 3 Intron ID from Table 3 ENST0000 0447742.6 NM_00108 1560ENSP00000 413417 NP_00107 5029 Protein coding EX2, EX3, EX4, EX5, EX6,EX8, EX9, EX10, EX11, EX12, EX13, EX16, EX18, EX19, EX20 INT2, INT3,INT4, INT5, INT8, INT9, INT10, INT11, INT12, INT15, INT17, INT18, INT19,INT20 ENST0000 0588522.5 ENSP00000 468013 Nonsense mediated decay EX37,EX38, EX39, EX40, EX41, EX42, EX43, EX44, EX45, EX46, EX47, EX48, EX49,EX50, EX51, EX52 INT37, INT38, INT39, INT40, INT41, INT42, INT43, INT44,INT45, INT46, INT47, INT48, INT49, INT50, INT51 ENST0000 0354227.9NM_00128 8766 ENSP00000 346168 NP_00127 5695 Protein coding EX2, EX3,EX4, EX5, EX6, EX8, EX9, EX10, EX11, EX16, EX19, EX26, EX27 INT2, INT3,INT4, INT5, INT8, INT9, INT10, INT15, INT18, INT19, INT26, INT27ENST0000 0291270.8 NM_00128 8764, NM_00440 9 ENSP00000 291270 NP_001275693, NP_00440 0 Protein coding EX2, EX3, EX4, EX5, EX6, EX7, EX8, EX9,EX10, EX11, EX12, EX13, EX15, EX16, EX17 INT2, INT3, INT4, INT5, INT6,INT7, INT8, INT9, INT10, INT11, INT12, INT14, INT15, INT16 ENST00000458663.6 NM_00108 1562 ENSP00000 401753 NP_00107 5031 Protein codingEX2, EX3, EX4, EX5, EX6, EX8, EX9, EX10, EX11, EX12, EX16, EX19, EX21,EX22, EX23 INT2, INT3, INT4, INT5, INT8, INT9, INT10, INT11, INT15,INT18, INT19, INT21, INT22, INT23 ENST0000 0600757.5 NM_00128 8765ENSP00000 472965 NP_00127 5694 Protein coding EX2, EX3, EX4, EX5, EX6,EX8, EX9, EX10, EX11, EX12, EX19, EX22, EX28, EX29 INT2, INT3, INT4,INT5, INT8, INT9, INT10, INT11, INT18, INT19, INT22, INT28, INT29ENST0000 0343373.8 NM_00108 1563 ENSP00000 345997 NP_00107 5032 Proteincoding EX1, EX2, EX3, EX4, EX5, EX6, EX7, EX8, EX9, EX10, EX11, EX12,EX13, EX14 INT1, INT2, INT3, INT4, INT5, INT6, INT7, INT8, INT9, INT10,INT11, INT12, INT13 ENST0000 0598180.1 Retained intron EX40, EX60, EX61INT58, INT59 ENST0000 0596686.5 Retained intron EX41, EX42, EX43, EX44,EX45, EX46, EX47, EX48, EX49, EX50, EX57, EX58, EX59 INT41, INT42,INT43, INT44, INT45, INT46, INT47, INT48, INT49, INT55, INT56, INT57ENST0000 0598272.1 Retained intron EX62 ENST0000 0596067.1 ENSP00000470192 Protein coding EX2, EX3, EX16, EX35, EX36 INT2, INT15, INT35,INT36 ENST0000 0599392.1 Retained intron EX67, EX68 INT64 ENST00000596920.1 Processed transcript EX42, EX43, EX55, EX56 INT42, INT53,INT54 ENST0000 0598191.5 Retained intron EX43, EX63, EX64 INT60, INT61ENST0000 0599002.1 Retained intron EX69, EX70 INT65 ENST0000 0595361.1Processed transcript EX53, EX54 INT52 ENST0000 0600370.1 Retained intronEX47, EX48, EX65, EX66 INT47, INT62, INT63 ENST0000 0593574.1 ENSP00000469220 Protein coding EX8, EX9, EX32, EX33, EX34 INT8, INT32, INT33,INT34 ENST0000 0597660.1 ENSP00000 471832 Protein coding EX10, EX11,EX30, EX31 INT10, INT30, INT31 ENST0000 0618091.4 ENSP00000 482746Protein coding EX2, EX3, EX4, EX5, EX6, EX8, EX9, EX10, EX11, EX12,EX16, EX19, EX21, EX22, EX24, EX25 INT2, INT3, INT4, INT5, INT8, INT9,INT10, INT11, INT15, INT18, INT19, INT21, INT22, INT24, INT25

DMPK has 1249 SNPs and the NCBI rs number and/or UniProt VAR number forthis DMPK gene are rs16939, rs498916, rs499726, rs517300, rs522004,rs522769, rs523577, rs527221 4, rs545759, rs546580, rs551399, rs557520,rs558180, rs572634, rs618370, rs618410, rs638400, rs638474, rs639363,rs639831, rs640685, rs657278, rs657640, rs659444, rs672348, rs689034,rs915915, rs1799894, rs2070737, rs2569769, rs2569770, rs2854335,rs2854336, rs2854337, rs2854338, rs3020642, rs4803854, rs7257693,rs7258468, rs10418454, rs10445573, rs11667776, rs11878503, rs12973612,rs34033836, rs34368222, rs34510782, rs34551308, rs34798952, rs34837201,rs34969749, rs35500073, rs35704059, rs36135587, rs36141801, rs56411618,rs59575950, rs61747614, rs71352289, rs71352290, rs73044281, rs73044286,rs73564346, rs73940321, rs74203721, rs74399432, rs74490545, rs74838358,rs74857182, rs74965490, rs75224362, rs75413061, rs75494688, rs75823295,rs75890421, rs76186545, rs76511276, rs76613387, rs76761831, rs77044979,rs77315725, rs77359036, rs77723844, rs77733323, rs77823890, rs78043518,rs78164767, rs78278813, rs78771765, rs79058930, rs79087711, rs79325850,rs79785531, rs80245764, rs111332235, rs111366282, rs111438673,rs111456489, rs111543904, rs111664796, rs111864835, rs112399907,rs112681257, rs112693590, rs112756864, rs112768469, rs113211813,rs113426968, rs113584565, rs113695200, rs113711374, rs114931926,rs115067015, rs115807657, rs115867412, rs116131451, rs116213648,rs138229648, rs138303455, rs138328610, rs138443073, rs138445739,rs138645023, rs138809535, rs138961255, rs138974608, rs139499893,rs139686338, rs139694707, rs139724884, rs140030942, rs140158944,rs140231303, rs140685882, rs140836596, rs140858879, rs141315683,rs141319926, rs141405839, rs141487873, rs141527316, rs141629958,rs142225348, rs142350523, rs142784795, rs142936719, rs142964084,rs143006643, rs143811850, rs144064078, rs144217170, rs144297716,rs144654756, rs144831190, rs144950565, rs145082393, rs145200358,rs145228732, rs145245565, rs145261129, rs145330026, rs145501208,rs146107996, rs146157132, rs146376068, rs146680240, rs146936052,rs147007284, rs147014815, rs147634105, rs147641100, rs147815859,rs147824333, rs148316122, rs148625275, rs149122951, rs149175283,rs149274125, rs149441245, rs149612963, rs149701607, rs149751203,rs149781731, rs149803658, rs149990515, rs150129351, rs150182960,rs150437533, rs150521628, rs150617093, rs150865718, rs150949514,rs151206095, rs151259364, rs181024723, rs181117943, rs181397555,rs181830960, rs182144573, rs182192093, rs182411745, rs183020005,rs183273843, rs183369470, rs183724763, rs183886672, rs183944228,rs184734237, rs185046805, rs185269445, rs186030887, rs186304356,rs186685924, rs186848593, rs186858574, rs187454872, rs187603639,rs188046657, rs188179179, rs188422169, rs188461283, rs188598689,rs188834458, rs189244368, rs189680884, rs190123449, rs190241849,rs190995682, rs191135249, rs191153689, rs191164250, rs191236114,rs191664249, rs191716386, rs191846663, rs192648726, rs192671827,rs192685724, rs193021388, rs193129899, rs193173337, rs199660819,rs199698181, rs199731275, rs199831687, rs199927858, rs199935282,rs200058479, rs200173713, rs200267773, rs200272101, rs200276159,rs200418173, rs200491028, rs200538327, rs200633144, rs200807652,rs200832423, rs200924908, rs201008625, rs201233585, rs201235465,rs201243969, rs201338891, rs201595882, rs201629532, rs201683145,rs201689719, rs201714957, rs201744975, rs201781936, rs202071119,rs202247396, rs367743734, rs368265414, rs368376507, rs368913582,rs368945420, rs369136836, rs369213664, rs369216416, rs369279468,rs369579996, rs369679086, rs369808315, rs369938946, rs369969645,rs370443174, rs370554703, rs370714807, rs370717859, rs370770361,rs370772195, rs370867347, rs370875536, rs370881189, rs371027164,rs371072226, rs371284143, rs371371836, rs371575564, rs371639503,rs371659096, rs371726191, rs371791113, rs371800676, rs371908326,rs371923244, rs372053831, rs372117801, rs372132101, rs372164940,rs372182032, rs372283432, rs372287704, rs372377505, rs372404548,rs372404776, rs372505372, rs372615381, rs372834428, rs370658995,rs370629839, rs370436340, rs370386108, rs370117370, rs370071817,rs370041130, rs369994896, rs369599562, rs369170331, rs368808213,rs368744145, rs368519758, rs368489171, rs368461139, rs368423827,rs202070753, rs372879396, rs372887904, rs372902369, rs372905696,rs373246151, rs373470824, rs373602912, rs373702492, rs373703629,rs373890653, rs373995039, rs374088757, rs374095905, rs374319207,rs374456013, rs374509696, rs374598653, rs374692384, rs374693463,rs374746286, rs374752093, rs374838493, rs374847549, rs374895405,rs375336589, rs375367726, rs375436452, rs375445989, rs375657646,rs375735536, rs375757084, rs375801671, rs375820845, rs375867547,rs376004763, rs376041280, rs376141910, rs376190478, rs376304022,rs376387926, rs376494290, rs376539020, rs376705114, rs376766483,rs376773457, rs376774997, rs377048280, rs377252920, rs377307057,rs377364945, rs377694144, rs377696477, rs377717953, rs377760466,rs527314968, rs527977407, rs528000342, rs528049631, rs528187884,rs528309659, rs528385615, rs528523331, rs528816876, rs529183986,rs529184674, rs529192626, rs529523895, rs529559215, rs529747471,rs529770568, rs529807413, rs530429994, rs530588467, rs530850230,rs530886873, rs530894415, rs531022115, rs531034368, rs531756450,rs531886633, rs531909370, rs532231379, rs532235846, rs532237384,rs532927349, rs533057934, rs533599401, rs533604882, rs533673483,rs533819750, rs533850058, rs533901178, rs533971633, rs534558623,rs534570520, rs534572562, rs534741898, rs534901766, rs534946812,rs535503559, rs535706406, rs535793935, rs536346070, rs536367261,rs536372592, rs536482577, rs536937476, rs536940737, rs537143850,rs537474918, rs537534284, rs537546070, rs537756502, rs538139992,rs538740165, rs538900872, rs538947784, rs539758310, rs540031135,rs540212663, rs540247396, rs540283241, rs540318330, rs540396129,rs540432774, rs540487664, rs541071823, rs541123797, rs541261983,rs541595046, rs541925402, rs542573366, rs542695091, rs542860247,rs543482651, rs544056738, rs544256614, rs544503512, rs545130086,rs545133449, rs545342019, rs545392455, rs545528789, rs545559067,rs545632314, rs545769272, rs545862530, rs546134855, rs546715112,rs546760773, rs546885773, rs547057811, rs547176945, rs547657762,rs547932881, rs548051873, rs548184612, rs548334598, rs548345202,rs548406200, rs548540888, rs548587032, rs548754043, rs549142572,rs549215033, rs549238614, rs549779754, rs550335928, rs550347923,rs550895847, rs550905850, rs550980476, rs551017682, rs551068247,rs551079890, rs551256079, rs551360062, rs551630953, rs551958417,rs552089682, rs552272866, rs552478694, rs553529394, rs553599209,rs553747237, rs553796756, rs554139617, rs554533315, rs554603776,rs554723850, rs554892086, rs554942363, rs554967270, rs555191515,rs555203925, rs555604484, rs556150348, rs556381398, rs556393555,rs556407652, rs556522505, rs556731727, rs556995262, rs557328143,rs557368237, rs557529836, rs557684528, rs557722032, rs558149662,rs558534447, rs558815378, rs559047228, rs559102204, rs559293117,rs559635432, rs560215560, rs560274727, rs560430710, rs560663357,rs560829987, rs560962451, rs561397709, rs561593638, rs562087330,rs562686918, rs562764391, rs562893010, rs563233712, rs563294621,rs563376175, rs563679367, rs563872372, rs564279709, rs565951155,rs566079768, rs566799097, rs567403565, rs567656507, rs568124260,rs568199378, rs568480287, rs568550546, rs570629415, rs570705950,rs571114682, rs571172627, rs571366543, rs572223902, rs572274820,rs572318377, rs572336424, rs572344877, rs572789035, rs572862323,rs572932741, rs572993435, rs573469092, rs573640511, rs573787396,rs573985824, rs574451407, rs574515658, rs574659132, rs574903423,rs574906809, rs574989745, rs575002841, rs575984462, rs576108279,rs576238440, rs576273903, rs576448654, rs576531655, rs577138224,rs570457920, rs570267997, rs569940022, rs569813020, rs569719355,rs569678890, rs569670872, rs569628728, rs569456096, rs569256146,rs569191457, rs568608046, rs565882456, rs565690376, rs565201970,rs565119809, rs562034960, rs562015011, rs561668197, rs577179879,rs577297413, rs577445591, rs577466943, rs577648783, rs577654202,rs577704005, rs577740076, rs577931918, rs578057735, rs745396904,rs745640651, rs745642406, rs745642787, rs745695704, rs745732334,rs745787405, rs745842483, rs745879777, rs745922025, rs745965249,rs746028393, rs746130533, rs746248791, rs746289800, rs746534953,rs746601368, rs746725048, rs746744094, rs746837426, rs746858804,rs746866067, rs747037860, rs747054349, rs747069302, rs747074970,rs747174968, rs747295381, rs747348407, rs747405436, rs747414929,rs747430642, rs747480403, rs747562157, rs747567839, rs747588213,rs747639886, rs747845597, rs747888241, rs747898634, rs747933477,rs747975834, rs747988487, rs748125970, rs748152953, rs748161989,rs748180973, rs748206543, rs748233374, rs748240647, rs748363348,rs748423751, rs748478867, rs748509070, rs748511524, rs748532051,rs748627080, rs748731304, rs748754867, rs748788537, rs748803132,rs748808453, rs748842067, rs748856470, rs748895029, rs749064317,rs749089111, rs749152970, rs749309049, rs749342663, rs749426150,rs749533341, rs749552724, rs749635716, rs749639132, rs749666645,rs749828765, rs749960267, rs749976267, rs749983369, rs750037893,rs750114122, rs750214189, rs750253660, rs750287221, rs750300600,rs750304884, rs750445251, rs750544307, rs750560808, rs750614096,rs750794715, rs750810488, rs750988317, rs751068497, rs751073998,rs751115299, rs751121702, rs751225267, rs751280117, rs751304727,rs751311764, rs751334898, rs751537685, rs751625776, rs751679078,rs751708764, rs751845196, rs752005444, rs752008622, rs752125836,rs752178847, rs752197228, rs752234867, rs752246818, rs752292803,rs752343582, rs752437441, rs752492337, rs752529012, rs752634390,rs752693597, rs752743750, rs752820666, rs752828192, rs752983975,rs753018860, rs753019364, rs753086131, rs753267971, rs753315592,rs753434777, rs753483995, rs753607433, rs753621189, rs753636314,rs753736470, rs753850894, rs753911237, rs753922780, rs753975059,rs754081706, rs754121701, rs754161431, rs754175256, rs754178866,rs754446129, rs754477746, rs754616373, rs754624984, rs754630789,rs754651761, rs754669888, rs754709046, rs754791976, rs754884245,rs754909474, rs754963585, rs754972369, rs755023780, rs755119412,rs755203771, rs755248058, rs755302835, rs755461013, rs755515708,rs755524201, rs755577294, rs755616825, rs755706868, rs755793415,rs755809532, rs755811609, rs755826884, rs755865044, rs755888149,rs755904408, rs755928451, rs756016441, rs756108365, rs756122292,rs756205218, rs756284189, rs756374992, rs756437038, rs756547906,rs756603673, rs756610907, rs756656828, rs756803484, rs756892589,rs756912200, rs756920572, rs756936150, rs756959604, rs757010986,rs757038408, rs757166836, rs757185658, rs757191303, rs757304220,rs757357412, rs757475031, rs757525597, rs757602115, rs757675176,rs757728098, rs757806074, rs757933338, rs757971036, rs758036606,rs758041683, rs758065350, rs758089399, rs758092645, rs758339461,rs758362923, rs758370954, rs758395409, rs758484481, rs758654664,rs758679761, rs758707798, rs758732755, rs758876872, rs758899830,rs758966003, rs758981137, rs759061105, rs759107509, rs759203349,rs759309228, rs759417388, rs759455411, rs759527747, rs759545957,rs759640975, rs759751429, rs759773909, rs759774926, rs759844893,rs759918150, rs759940583, rs759945808, rs760073904, rs760107585,rs760240414, rs760386570, rs760454262, rs760599845, rs760600703,rs760653625, rs760708972, rs760881334, rs760956137, rs761002462,rs761077797, rs761086754, rs761137550, rs761241092, rs761250462,rs761275554, rs761285661, rs761361885, rs761384159, rs761415826,rs761618825, rs761649903, rs761652471, rs761707572, rs761736775,rs761744296, rs761832870, rs761882172, rs761884259, rs762094656,rs762280354, rs762331478, rs762427243, rs762450145, rs762467984,rs762562873, rs762721322, rs762730157, rs762774180, rs762784046,rs762835443, rs762915357, rs763021104, rs763083772, rs763094385,rs763102565, rs763177539, rs763333603, rs763358330, rs763412184,rs763442319, rs763476430, rs763497515, rs763738699, rs763784457,rs763926129, rs764039581, rs764041434, rs764051493, rs764094650,rs764146518, rs764218817, rs762230984, rs760130544, rs759063685,rs760175683, rs764372949, rs764399605, rs764428073, rs764488056,rs764517414, rs764541567, rs764722784, rs764855500, rs764870708,rs764878781, rs764925151, rs764957507, rs765043098, rs765164337,rs765166457, rs765177343, rs765190599, rs765438132, rs765451387,rs765503777, rs765558854, rs765628468, rs765691118, rs765751328,rs765754023, rs765755431, rs765918291, rs766012197, rs766021138,rs766093732, rs766138774, rs766151412, rs766274584, rs766282916,rs766435666, rs766485074, rs766507812, rs766547162, rs766622379,rs766818029, rs767095031, rs767174205, rs767229336, rs767282165,rs767305569, rs767322336, rs767453806, rs767470857, rs767476529,rs767498563, rs767615534, rs767616508, rs767706751, rs767793835,rs767816157, rs767864367, rs767991260, rs767999474, rs768038880,rs768148361, rs768189290, rs768263033, rs768279580, rs768362613,rs768556739, rs768559586, rs768671280, rs768779192, rs768812283,rs768832274, rs768834265, rs768887265, rs769190636, rs769213954,rs769239379, rs769267041, rs769279359, rs769307302, rs769357491,rs769358934, rs769373576, rs769410837, rs769513377, rs769572842,rs769610342, rs769618931, rs769665642, rs769700516, rs770005245,rs770047523, rs770110893, rs770112203, rs770140484, rs770198290,rs770389641, rs770405145, rs770443048, rs770502257, rs770590594,rs770656856, rs770724824, rs770968394, rs771057092, rs771063469,rs771066769, rs771089695, rs771093221, rs771161939, rs771182555,rs771214913, rs771259599, rs771358616, rs771380204, rs771438087,rs771444624, rs771472462, rs771536499, rs771541491, rs771591816,rs771592769, rs771700662, rs771735401, rs771937879, rs772025839,rs772059693, rs772131296, rs772134866, rs772207631, rs772259297,rs772262669, rs772267314, rs772315750, rs772363177, rs772444621,rs772454721, rs772493086, rs772637316, rs772659173, rs772684221,rs772688520, rs772727496, rs772749279, rs772869173, rs772915546,rs772917754, rs772944851, rs773115494, rs773149416, rs773176765,rs773201886, rs773217636, rs773246625, rs773315981, rs773325557,rs773427392, rs773427732, rs773525720, rs773619992, rs773706825,rs773757842, rs773760078, rs773761593, rs773845103, rs773849625,rs773866262, rs773936389, rs774007631, rs774011333, rs774037322,rs774041974, rs774256278, rs774278273, rs774307845, rs774336633,rs774384090, rs774479026, rs774500030, rs774662606, rs774774759,rs774809286, rs774829985, rs774862968, rs774925987, rs775014801,rs775118554, rs775317730, rs775395773, rs775463380, rs775505956,rs775509391, rs775626849, rs775684002, rs775811905, rs775843620,rs775911829, rs775956604, rs775989938, rs776031050, rs776093870,rs776670683, rs776686686, rs776793721, rs776994468, rs777008895,rs777077770, rs777107813, rs777189457, rs777284682, rs777398172,rs777469471, rs777489712, rs777520924, rs777531284, rs777582664,rs777659435, rs777875351, rs777988066, rs777996567, rs778048098,rs778049248, rs778249103, rs778302103, rs778309745, rs778365025,rs778431182, rs778455449, rs778635591, rs778669160, rs778687006,rs778777182, rs778806268, rs778994221, rs779008833, rs779163636,rs779195244, rs779230589, rs779683741, rs779695594, rs779781291,rs779792982, rs779889773, rs779923404, rs780074257, rs780103378,rs780187592, rs780263576, rs780266570, rs780346478, rs780386907,rs780659624, rs780801121, rs780833135, rs780833366, rs780860038,rs780885997, rs780897613, rs780962756, rs781005180, rs781010588,rs781379548, rs781430944, rs781435746, rs781436323, rs781600675,rs781600764, rs781678068, rs781734640, VAR_040452, rs781095880,rs781085771, rs780653085, rs780560028, rs780433829, rs780415848,rs780397803, rs779534958, rs779481949, rs779440487, rs779252472,rs779126423, rs778926101, rs777783703, rs776849518, rs776764650,rs776619322, rs776266757, rs776234804, rs775977785, rs775976010,rs775964614, rs775571843, rs774470818, rs774435498, rs781283859,rs781234107, and rs781155031.

In one example, the guide RNA used in the present disclosure maycomprise at least one 20 nucleotide (nt) target nucleic acid sequencelisted in Table 5. Provided in Table 5 are the gene symbol and thesequence identifier of the gene (Gene SEQ ID NO), the gene sequenceincluding 1-5 kilobase pairs upstream and/or downstream of the targetgene (Extended Gene SEQ ID NO), and the 20 nt target nucleic acidsequence (20 nt Target Sequence SEQ ID NO). In the sequence listing therespective target gene, the strand for targeting the gene (noted by a(+) strand or (-) strand in the sequence listing), the associated PAMtype and the PAM sequence are described for each of the 20 nt targetnucleic acid sequences (SEQ ID NO: 5305-20697). It is understood in theart that the spacer sequence, where “T” is “U,” may be an RNA sequencecorresponding to the 20 nt sequences listed in Table 5.

TABLE 5 Nucleic Acid Sequences Gene Symbol Gene SEQ ID NO Extended GeneSEQ ID NO 20 nt Target Sequence SEQ ID NO DMPK 5303 5304 5305-20697

In one example, the guide RNA used in the disclosure may comprise atleast one spacer sequence that, where “T” is “U”, may be an RNA sequencecorresponding to a 20 nucleotide (nt) target sequence such as, but notlimited to, any of SEQ ID NO: 5305-20697.

In one example, the guide RNA used in the disclosure may comprise atleast one spacer sequence which, where “T” is “U,” is an RNA sequencecorresponding to the 20 nt sequences such as, but not limited to, any ofSEQ ID NO: 5305-20697.

In one example, a guide RNA may comprise a 20 nucleotide (nt) targetnucleic acid sequence associated with the PAM type such as, but notlimited to, NAAAAC, NNAGAAW, NNGRRT, NNNNGHTT, NRG, or YTN. As anon-limiting example, the 20 nt target nucleic acid sequence for aspecific target gene and a specific PAM type may be, where “T” is “U,”the RNA sequence corresponding to any one of the 20 nt nucleic acidsequences in Table 6.

TABLE 6 Nucleic Acid Sequences by PAM Type Gene Symbol PAM: NAAAAC PAM:NNAGAAW PAM: NNGRRT PAM: NNNNGHTT PAM: NRG PAM: YTN 20 nt Target NucleicAcid SEQ ID NO 20 nt Target Nucleic Acid SEQ ID NO 20 nt Target NucleicAcid SEQ ID NO 20 nt Target Nucleic Acid SEQ ID NO 20 nt Target NucleicAcid SEQ ID NO 20 nt Target Nucleic Acid SEQ ID NO DMPK 5305-53325333-5399 5400-6048 6049-6367 6368-15236 15237-20697

In one example, a guide RNA may comprise a 20 nucleotide (nt) targetnucleic acid sequence associated with the YTN PAM type. As anon-limiting example, the 20 nt target nucleic acid sequence for aspecific target gene may comprise a 20 nt core sequence where the 20 ntcore sequence, where “T” is “U,” may be the RNA sequence correspondingto SEQ ID NO: 15237-20697. As another non-limiting example, the 20 nttarget nucleic acid sequence for a specific target gene may comprise acore sequence where the core sequence, where “T” is “U,” may be afragment, segment or region of the RNA sequence corresponding to any ofSEQ ID NO: 15237-20697.

VI. Other Therapeutic Approaches

Gene editing can be conducted using nucleases engineered to targetspecific sequences. To date there are four major types of nucleases:meganucleases and their derivatives, zinc finger nucleases (ZFNs),transcription activator like effector nucleases (TALENs), andCRISPR-Cas9 nuclease systems. The nuclease platforms vary in difficultyof design, targeting density and mode of action, particularly as thespecificity of ZFNs and TALENs is through protein-DNA interactions,while RNA-DNA interactions primarily guide Cas9.

CRISPR endonucleases, such as Cas9, can be used in the methods of thepresent disclosure. However, the teachings described herein, such astherapeutic target sites, could be applied to other forms ofendonucleases, such as ZFNs, TALENs, HEs, or MegaTALs, or usingcombinations of nucleases. However, in order to apply the teachings ofthe present disclosure to such endonucleases, one would need to, amongother things, engineer proteins directed to the specific target sites.

Additional binding domains can be fused to the Cas9 protein to increasespecificity. The target sites of these constructs would map to theidentified gRNA specified site, but would require additional bindingmotifs, such as for a zinc finger domain. In the case of Mega-TAL, ameganuclease can be fused to a TALE DNA-binding domain. The meganucleasedomain can increase specificity and provide the cleavage. Similarly,inactivated or dead Cas9 (dCas9) can be fused to a cleavage domain andrequire the sgRNA/Cas9 target site and adjacent binding site for thefused DNA-binding domain. This likely would require some proteinengineering of the dCas9, in addition to the catalytic inactivation, todecrease binding without the additional binding site.

Zinc Finger Nucleases

Zinc finger nucleases (ZFNs) are modular proteins comprised of anengineered zinc finger DNA binding domain linked to the catalytic domainof the type II endonuclease FokI. Because FokI functions only as adimer, a pair of ZFNs must be engineered to bind to cognate target“half-site” sequences on opposite DNA strands and with precise spacingbetween them to enable the catalytically active FokI dimer to form. Upondimerization of the FokI domain, which itself has no sequencespecificity per se, a DNA double-strand break is generated between theZFN half-sites as the initiating step in genome editing.

The DNA binding domain of each ZFN is typically comprised of 3-6 zincfingers of the abundant Cys2-His2 architecture, with each fingerprimarily recognizing a triplet of nucleotides on one strand of thetarget DNA sequence, although cross-strand interaction with a fourthnucleotide also can be important. Alteration of the amino acids of afinger in positions that make key contacts with the DNA alters thesequence specificity of a given finger. Thus, a four-finger zinc fingerprotein will selectively recognize a 12 bp target sequence, where thetarget sequence is a composite of the triplet preferences contributed byeach finger, although triplet preference can be influenced to varyingdegrees by neighboring fingers. An important aspect of ZFNs is that theycan be readily re-targeted to almost any genomic address simply bymodifying individual fingers, although considerable expertise isrequired to do this well. In most applications of ZFNs, proteins of 4-6fingers are used, recognizing 12-18 bp respectively. Hence, a pair ofZFNs will typically recognize a combined target sequence of 24-36 bp,not including the typical 5-7 bp spacer between half-sites. The bindingsites can be separated further with larger spacers, including 15-17 bp.A target sequence of this length is likely to be unique in the humangenome, assuming repetitive sequences or gene homologs are excludedduring the design process. Nevertheless, the ZFN protein-DNAinteractions are not absolute in their specificity so off-target bindingand cleavage events do occur, either as a heterodimer between the twoZFNs, or as a homodimer of one or the other of the ZFNs. The latterpossibility has been effectively eliminated by engineering thedimerization interface of the FokI domain to create “plus” and “minus”variants, also known as obligate heterodimer variants, which can onlydimerize with each other, and not with themselves. Forcing the obligateheterodimer prevents formation of the homodimer. This has greatlyenhanced specificity of ZFNs, as well as any other nuclease that adoptsthese FokI variants.

A variety of ZFN-based systems have been described in the art,modifications thereof are regularly reported, and numerous referencesdescribe rules and parameters that are used to guide the design of ZFNs;see, e.g., Segal et al., Proc Natl Acad Sci USA 96(6):2758-63 (1999);Dreier B et al., J Mol Biol. 303(4):489-502 (2000); Liu Q et al., J BiolChem. 277(6):3850-6 (2002); Dreier et al., J Biol Chem 280(42):35588-97(2005); and Dreier et al., J Biol Chem. 276(31):29466-78 (2001).

Transcription Activator-Like Effector Nucleases (TALENs)

TALENs represent another format of modular nucleases whereby, as withZFNs, an engineered DNA binding domain is linked to the FokI nucleasedomain, and a pair of TALENs operate in tandem to achieve targeted DNAcleavage. The major difference from ZFNs is the nature of the DNAbinding domain and the associated target DNA sequence recognitionproperties. The TALEN DNA binding domain derives from TALE proteins,which were originally described in the plant bacterial pathogenXanthomonas sp. TALEs are comprised of tandem arrays of 33-35 amino acidrepeats, with each repeat recognizing a single base pair in the targetDNA sequence that is typically up to 20 bp in length, giving a totaltarget sequence length of up to 40 bp. Nucleotide specificity of eachrepeat is determined by the repeat variable diresidue (RVD), whichincludes just two amino acids at positions 12 and 13. The bases guanine,adenine, cytosine and thymine are predominantly recognized by the fourRVDs: Asn-Asn, Asn-Ile, His-Asp and Asn-Gly, respectively. Thisconstitutes a much simpler recognition code than for zinc fingers, andthus represents an advantage over the latter for nuclease design.Nevertheless, as with ZFNs, the protein-DNA interactions of TALENs arenot absolute in their specificity, and TALENs have also benefitted fromthe use of obligate heterodimer variants of the FokI domain to reduceoff-target activity.

Additional variants of the FokI domain have been created that aredeactivated in their catalytic function. If one half of either a TALENor a ZFN pair contains an inactive FokI domain, then only single-strandDNA cleavage (nicking) will occur at the target site, rather than a DSB.The outcome is comparable to the use of CRISPR/Cas9 or CRISPR/Cpf1“nickase” mutants in which one of the Cas9 cleavage domains has beendeactivated. DNA nicks can be used to drive genome editing by HDR, butat lower efficiency than with a DSB. The main benefit is that off-targetnicks are quickly and accurately repaired, unlike the DSB, which isprone to NHEJ-mediated mis-repair.

A variety of TALEN-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., Boch, Science326(5959): 1509-12 (2009); Mak et al., Science 335(6069):716-9 (2012);and Moscou et al., Science 326(5959):1501 (2009). The use of TALENsbased on the “Golden Gate” platform, or cloning scheme, has beendescribed by multiple groups; see, e.g., Cermak et al., Nucleic AcidsRes. 39(12):e82 (2011); Li et al., Nucleic Acids Res.39(14):6315-25(2011); Weber et al., PLoS One. 6(2):e16765 (2011); Wanget al., J Genet Genomics 41(6):339-47, Epub 2014 May 17 (2014); andCermak T et al., Methods Mol Biol. 1239:133-59 (2015).

Homing Endonucleases

Homing endonucleases (HEs) are sequence-specific endonucleases that havelong recognition sequences (14-44 base pairs) and cleave DNA with highspecificity - often at sites unique in the genome. There are at leastsix known families of HEs as classified by their structure, includingGIY-YIG, His-Cis box, H-N-H, PD-(D/E)xK, and Vsr-like that are derivedfrom a broad range of hosts, including eukarya, protists, bacteria,archaea, cyanobacteria and phage. As with ZFNs and TALENs, HEs can beused to create a DSB at a target locus as the initial step in genomeediting. In addition, some natural and engineered HEs cut only a singlestrand of DNA, thereby functioning as site-specific nickases. The largetarget sequence of HEs and the specificity that they offer have madethem attractive candidates to create site-specific DSBs.

A variety of HE-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., the reviews bySteentoft et al., Glycobiology 24(8):663-80 (2014); Belfort andBonocora, Methods Mol Biol. 1123:1-26 (2014); Hafez and Hausner, Genome55(8):553-69 (2012); and references cited therein.

MegaTAL / Tev-mTALEN / MegaTev

As further examples of hybrid nucleases, the MegaTAL platform andTev-mTALEN platform use a fusion of TALE DNA binding domains andcatalytically active HEs, taking advantage of both the tunable DNAbinding and specificity of the TALE, as well as the cleavage sequencespecificity of the HE; see, e.g., Boissel et al., NAR 42: 2591-2601(2014); Kleinstiver et al., G3 4:1155-65 (2014); and Boissel andScharenberg, Methods Mol. Biol. 1239: 171-96 (2015).

In a further variation, the MegaTev architecture is the fusion of ameganuclease (Mega) with the nuclease domain derived from the GIY-YIGhoming endonuclease I-TevI (Tev). The two active sites are positioned~30 bp apart on a DNA substrate and generate two DSBs withnon-compatible cohesive ends; see, e.g., Wolfs et al., NAR 42, 8816-29(2014). It is anticipated that other combinations of existingnuclease-based approaches will evolve and be useful in achieving thetargeted genome modifications described herein.

dCas9-FokIor dCpf1-Fok1 and Other Nucleases

Combining the structural and functional properties of the nucleaseplatforms described above offers a further approach to genome editingthat can potentially overcome some of the inherent deficiencies. As anexample, the CRISPR genome editing system typically uses a single Cas9endonuclease to create a DSB. The specificity of targeting is driven bya 20 or 24 nucleotide sequence in the guide RNA that undergoesWatson-Crick base-pairing with the target DNA (plus an additional 2bases in the adjacent NAG or NGG PAM sequence in the case of Cas9 fromS. pyogenes). Such a sequence is long enough to be unique in the humangenome, however, the specificity of the RNA/DNA interaction is notabsolute, with significant promiscuity sometimes tolerated, particularlyin the 5’ half of the target sequence, effectively reducing the numberof bases that drive specificity. One solution to this has been tocompletely deactivate the Cas9 or Cpfl catalytic function – retainingonly the RNA-guided DNA binding function – and instead fusing a FokIdomain to the deactivated Cas9; see, e.g., Tsai et al., Nature Biotech32: 569-76 (2014); and Guilinger et al., Nature Biotech. 32: 577-82(2014). Because FokI must dimerize to become catalytically active, twoguide RNAs are required to tether two FokI fusions in close proximity toform the dimer and cleave DNA. This essentially doubles the number ofbases in the combined target sites, thereby increasing the stringency oftargeting by CRISPR-based systems.

As further example, fusion of the TALE DNA binding domain to acatalytically active HE, such as I-TevI, takes advantage of both thetunable DNA binding and specificity of the TALE, as well as the cleavagesequence specificity of I-TevI, with the expectation that off-targetcleavage can be further reduced.

VII. Kits

The present disclosure provides kits for carrying out the methodsdescribed herein. A kit can include one or more of a genome-targetingnucleic acid, a polynucleotide encoding a genome-targeting nucleic acid,a site-directed polypeptide, a polynucleotide encoding a site-directedpolypeptide, and/or any nucleic acid or proteinaceous molecule necessaryto carry out the aspects of the methods described herein, or anycombination thereof.

A kit can comprise: (1) a vector comprising a nucleotide sequenceencoding a genome-targeting nucleic acid, (2) the site-directedpolypeptide or a vector comprising a nucleotide sequence encoding thesite-directed polypeptide, and (3) a reagent for reconstitution and/ordilution of the vector(s) and or polypeptide.

A kit can comprise: (1) a vector comprising (i) a nucleotide sequenceencoding a genome-targeting nucleic acid, and (ii) a nucleotide sequenceencoding the site-directed polypeptide; and (2) a reagent forreconstitution and/or dilution of the vector.

In any of the above kits, the kit can comprise a single-molecule guidegenome-targeting nucleic acid. In any of the above kits, the kit cancomprise a double-molecule genome-targeting nucleic acid. In any of theabove kits, the kit can comprise two or more double-molecule guides orsingle-molecule guides. The kits can comprise a vector that encodes thenucleic acid targeting nucleic acid.

In any of the above kits, the kit can further comprise a polynucleotideto be inserted to effect the desired genetic modification.

Components of a kit can be in separate containers, or combined in asingle container.

Any kit described above can further comprise one or more additionalreagents, where such additional reagents are selected from a buffer, abuffer for introducing a polypeptide or polynucleotide into a cell, awash buffer, a control reagent, a control vector, a control RNApolynucleotide, a reagent for in vitro production of the polypeptidefrom DNA, adaptors for sequencing and the like. A buffer can be astabilization buffer, a reconstituting buffer, a diluting buffer, or thelike. A kit can also comprise one or more components that can be used tofacilitate or enhance the on-target binding or the cleavage of DNA bythe endonuclease, or improve the specificity of targeting.

In addition to the above-mentioned components, a kit can furthercomprise instructions for using the components of the kit to practicethe methods. The instructions for practicing the methods can be recordedon a suitable recording medium. For example, the instructions can beprinted on a substrate, such as paper or plastic, etc. The instructionscan be present in the kits as a package insert, in the labeling of thecontainer of the kit or components thereof (i.e., associated with thepackaging or subpackaging), etc. The instructions can be present as anelectronic storage data file present on a suitable computer readablestorage medium, e.g. CD-ROM, diskette, flash drive, etc. In someinstances, the actual instructions are not present in the kit, but meansfor obtaining the instructions from a remote source (e.g. via theInternet), can be provided. An example of this case is a kit thatcomprises a web address where the instructions can be viewed and/or fromwhich the instructions can be downloaded. As with the instructions, thismeans for obtaining the instructions can be recorded on a suitablesubstrate.

VIII. Specific Methods and Compositions of the Invention

Accordingly, the present disclosure relates in particular to thefollowing non-limiting methods according to the disclosure: in a firstmethod, Method 1, the present disclosure provides a method for editing aDystrophia Myotonica-Protein Kinase (DMPK) gene in a cell by genomeediting comprising: introducing into the cell one or moredeoxyribonucleic acid (DNA) endonucleases to effect one or moresingle-strand breaks (SSBs) or double-strand breaks (DSBs) within ornear the DMPK gene that results in permanent deletion of the expandedtrinucleotide repeat or replacement of one or more nucleotide bases, orone or more exons and/or introns within or near the DMPK gene, therebyrestoring the DMPK gene function.

In another method, Method 2, the present disclosure provides a methodfor editing a Dystrophia Myotonica-Protein Kinase (DMPK) gene in a humancell by genome editing comprising: introducing into the human cell oneor more deoxyribonucleic acid (DNA) endonucleases to effect one or moresingle-strand breaks (SSBs) or double-strand breaks (DSBs) within ornear the DMPK gene or DMPK regulatory elements that results in one ormore permanent insertion, deletion or mutation of at least onenucleotide within or near the DMPK gene, thereby reducing or eliminatingthe expression or function of aberrant DMPK gene products.

In another method, Method 3, the present disclosure provides an ex vivomethod for treating a patient having a DMPK related condition ordisorder comprising: isolating a muscle cell or muscle precursor cellfrom a patient; editing within or near a Dystrophia Myotonica-ProteinKinase (DMPK) gene or other DNA sequences that encode regulatoryelements of the DMPK gene of the muscle cell or muscle precursor cell;and implanting the genome-edited muscle cell or muscle precursor cellinto the patient.

In another method, Method 4, the present disclosure provides the methodof Method 3, wherein the editing step comprises introducing into themuscle cell or muscle precursor cell one or more deoxyribonucleic acid(DNA) endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the DMPK gene that results inpermanent deletion of the expanded trinucleotide repeat or replacementof one or more nucleotide bases, or one or more exons and/or intronswithin or near the DMPK gene, thereby restoring the DMPK gene function.

In another method, Method 5, the present disclosure provides the methodof Method 3, wherein the editing step comprises introducing into themuscle cell or muscle precursor cell one or more deoxyribonucleic acid(DNA) endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the DMPK gene or DMPKregulatory elements that results in one or more permanent insertion,deletion or mutation of at least one nucleotide within or near the DMPKgene, thereby reducing or eliminating the expression or function ofaberrant DMPK gene products.

In another method, Method 6, the present disclosure provides the methodof any of Methods 3-5, wherein the muscle cell is a skeletal musclecell.

In another method, Method 7, the present disclosure provides the methodof any of Methods 3-5, wherein the muscle cell is a smooth muscle cell.

In another method, Method 8, the present disclosure provides the methodof any of Methods 3-5, wherein the muscle cell is a cardiac muscle cell.

In another method, Method 9, the present disclosure provides an ex vivomethod for treating a patient having a DMPK related condition ordisorder comprising: creating a patient specific induced pluripotentstem cell (iPSC); editing within or near a Dystrophia Myotonica-ProteinKinase (DMPK) gene or other DNA sequences that encode regulatoryelements of the DMPK gene of the iPSC; differentiating the genome-editediPSC into a skeletal muscle cell, a smooth muscle cell, a cardiac musclecell or a Pax7+ myocyte progenitor cell; and implanting the skeletalmuscle cell, smooth muscle cell, cardiac muscle cell or Pax7+ myocyteprogenitor cell into the patient.

In another method, Method 10, the present disclosure provides the methodof Method 9, wherein the editing step comprises introducing into theiPSC one or more deoxyribonucleic acid (DNA) endonucleases to effect oneor more single-strand breaks (SSBs) or double-strand breaks (DSBs)within or near the DMPK gene that results in permanent deletion of theexpanded trinucleotide repeat or replacement of one or more nucleotidebases, or one or more exons and/or introns within or near the DMPK gene,thereby restoring the DMPK gene function.

In another method, Method 11, the present disclosure provides the methodof Method 9, wherein the editing step comprises introducing into theiPSC one or more deoxyribonucleic acid (DNA) endonucleases to effect oneor more single-strand breaks (SSBs) or double-strand breaks (DSBs)within or near the DMPK gene or DMPK regulatory elements that results inone or more permanent insertion, deletion or mutation of at least onenucleotide within or near the DMPK gene, thereby reducing or eliminatingthe expression or function of aberrant DMPK gene products.

In another method, Method 12, the present disclosure provides an ex vivomethod for treating a patient having a DMPK related condition ordisorder comprising: isolating a mesenchymal stem cell from the patient;editing within or near a Dystrophia Myotonica-Protein Kinase (DMPK) geneor other DNA sequences that encode regulatory elements of the DMPK geneof the mesenchymal stem cell; differentiating the genome-editedmesenchymal stem cell into a skeletal muscle cell, a smooth muscle cell,a cardiac muscle cell or a Pax7+ myocyte progenitor cell; and implantingthe skeletal muscle cell, smooth muscle cell, cardiac muscle cell orPax7+ myocyte progenitor cell into the patient.

In another method, Method 13, the present disclosure provides the methodof Method 12, wherein the editing step comprises introducing into themesenchymal stem cell one or more deoxyribonucleic acid (DNA)endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the DMPK gene that results inpermanent deletion of the expanded trinucleotide repeat or replacementof one or more nucleotide bases, or one or more exons and/or intronswithin or near the DMPK gene, thereby restoring the DMPK gene function.

In another method, Method 14, the present disclosure provides the methodof Method 12, wherein the editing step comprises introducing into themesenchymal stem cell one or more deoxyribonucleic acid (DNA)endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the DMPK gene or DMPKregulatory elements that results in one or more permanent insertion,deletion or mutation of at least one nucleotide within or near the DMPKgene, thereby reducing or eliminating the expression or function ofaberrant DMPK gene products.

In another method, Method 15, the present disclosure provides an in vivomethod for treating a patient with a DMPK related disorder comprisingediting the Dystrophia Myotonica-Protein Kinase (DMPK) gene in a cell ofthe patient.

In another method, Method 16, the present disclosure provides the methodof Method 15, wherein the editing step comprises introducing into thecell one or more deoxyribonucleic acid (DNA) endonucleases to effect oneor more single-strand breaks (SSBs) or double-strand breaks (DSBs)within or near the DMPK gene that results in permanent deletion of theexpanded trinucleotide repeat or replacement of one or more nucleotidebases, or one or more exons and/or introns within or near the DMPK gene,thereby restoring the DMPK gene function.

In another method, Method 17, the present disclosure provides the methodof Method 15, wherein the editing step comprises introducing into thecell one or more deoxyribonucleic acid (DNA) endonucleases to effect oneor more single-strand breaks (SSBs) or double-strand breaks (DSBs)within or near the DMPK gene or DMPK regulatory elements that results inone or more permanent insertion, deletion or mutation of at least onenucleotide within or near the DMPK gene, thereby reducing or eliminatingthe expression or function of aberrant DMPK gene products.

In another method, Method 18, the present disclosure provides the methodof any one of Methods 15-17, wherein the cell is a muscle cell or muscleprecursor cell.

In another method, Method 19, the present disclosure provides the methodof Method 18, wherein the muscle cell is a skeletal muscle cell.

In another method, Method 20, the present disclosure provides the methodof Method 18, wherein the muscle cell is a smooth muscle cell.

In another method, Method 21, the present disclosure provides the methodof Method 18, wherein the muscle cell is a cardiac muscle cell.

In another method, Method 22, the present disclosure provides the methodof any one of Methods 18-20, wherein the one or more deoxyribonucleicacid (DNA) endonuclease is delivered to the muscle cell or muscleprecursor cell by local injection into the desired muscle.

In another method, Method 23, the present disclosure provides a methodof altering the contiguous genomic sequence of a DMPK gene in a cellcomprising contacting the cell with one or more deoxyribonucleic acid(DNA) endonuclease to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs).

In another method, Method 24, the present disclosure provides the methodof Method 23, wherein the alteration of the contiguous genomic sequenceoccurs in one or more exons of the DMPK gene.

In another method, Method 25, the present disclosure provides the methodof Method 24, wherein the alteration of the contiguous genomic sequenceoccurs in the 3’ untranslated region (UTR) of the DMPK gene.

In another method, Method 26, the present disclosure provides the methodof any one of Methods 1-25, wherein the one or more deoxyribonucleicacid (DNA) endonuclease is selected from any of those sequences in SEQID NOs: 1-620, and variants having at least 90% homology to any of thosesequences disclosed in SEQ ID NOs: 1-620.

In another method, Method 27, the present disclosure provides the methodof Method 26, wherein the one or more deoxyribonucleic acid (DNA)endonuclease is one or more protein or polypeptide.

In another method, Method 28, the present disclosure provides the methodof Method 26, wherein the one or more deoxyribonucleic acid (DNA)endonuclease is one or more polynucleotide encoding the one or more DNAendonuclease.

In another method, Method 29, the present disclosure provides the methodof Method 28, wherein the one or more deoxyribonucleic acid (DNA)endonuclease is one or more ribonucleic acid (RNA) encoding the one ormore DNA endonuclease.

In another method, Method 30, the present disclosure provides the methodof Method 29, wherein the one or more ribonucleic acid (RNA) is one ormore chemically modified RNA.

In another method, Method 31, the present disclosure provides the methodof Method 30, wherein the one or more ribonucleic acid (RNA) ischemically modified in the coding region.

In another method, Method 32, the present disclosure provides the methodof any one of Methods 28-31, wherein the one or more polynucleotide orone or more ribonucleic acid (RNA) is codon optimized.

In another method, Method 33, the present disclosure provides the methodof any one of Methods 1-32, wherein the method further comprisesintroducing one or more gRNA or one or more sgRNA.

In another method, Method 34, the present disclosure provides the methodof Method 33, wherein the one or more gRNA or one or more sgRNAcomprises a spacer sequence that is complementary to a sequence withinor near the expanded trinucleotide repeat in the DMPK gene.

In another method, Method 35, the present disclosure provides the methodof Method 33, wherein the one or more gRNA or one or more sgRNAcomprises a spacer sequence that is complementary to a DNA sequencewithin or near the DMPK gene.

In another method, Method 36, the present disclosure provides the methodof Method 33, wherein the one or more gRNA or one or more sgRNAcomprises a spacer sequence that is complementary to a sequence flankingthe DMPK gene or other sequence that encodes a regulatory element of theDMPK gene.

In another method, Method 37, the present disclosure provides, thepresent disclosure provides the method of any one of Methods 33-36,wherein the one or more gRNA or one or more sgRNA is chemicallymodified.

In another method, Method 38, the present disclosure provides the methodof any one of Methods 33-37, wherein the one or more gRNA or one or moresgRNA is pre-complexed with the one or more deoxyribonucleic acid (DNA)endonuclease.

In another method, Method 39, the present disclosure provides the methodof Method 38, wherein the pre-complexing involves a covalent attachmentof the one or more gRNA or one or more sgRNA to the one or moredeoxyribonucleic acid (DNA) endonuclease.

In another method, Method 40, the present disclosure provides the methodof any one of Methods 26-39, wherein the one or more deoxyribonucleicacid (DNA) endonuclease is formulated in a liposome or lipidnanoparticle.

In another method, Method 41, the present disclosure provides the methodof any one of Methods 33-39, wherein the one or more deoxyribonucleicacid (DNA) endonuclease is formulated in a liposome or a lipidnanoparticle which also comprises the one or more gRNA or one or moresgRNA.

In another method, Method 42, the present disclosure provides the methodof any one of Methods 26, or 33-36, wherein the one or moredeoxyribonucleic acid (DNA) endonuclease is encoded in an AAV vectorparticle.

In another method, Method 43, the present disclosure provides the methodof any one of Methods 33-36, wherein the one or more gRNA or one or moresgRNA is encoded in an AAV vector particle.

In another method, Method 44, the present disclosure provides the methodof any one of Methods 33-36, wherein the one or more deoxyribonucleicacid (DNA) endonuclease is encoded in an AAV vector particle which alsoencodes the one or more gRNA or one or more sgRNA.

In another method, Method 45, the present disclosure provides the methodof any one of Methods 42-44, whereing the AAV vector particle isselected from the group consisting of any of those disclosed in SEQ IDNOs: 4734-5302 and Table 2.

In another method, Method 46, the present disclosure provides the methodof any of Methods 1-45, wherein the method further comprises introducinginto the cell a donor template comprising at least a portion of thewild-type DMPK gene.

In another method, Method 47, the present disclosure provides the methodof Method 46, wherein the at least a portion of the wild-type DMPK genecomprises one or more sequences selected from the group consisting of: aDMPK exon, a DMPK intron, and a sequence comprising an exon:intronjunction of DMPK.

In another method, Method 48, the present disclosure provides the methodof any one of Methods 46-47, wherein the donor template compriseshomologous arms to the genomic locus of the DMPK gene.

In another method, Method 49, the present disclosure provides the methodof any one of Methods 46-48, wherein the donor template is either asingle or double stranded polynucleotide.

In another method, Method 50, the present disclosure provides the methodof any one of Methods 46-49, wherein the donor template is encoded in anAAV vector particle.

In another method, Method 51, the present disclosure provides the methodof Method 50, wherein the AAV vector particle is selected from the groupconsisting of any of those disclosed in SEQ ID NOs: 4734-5302 and Table2.

In another method, Method 52, the present disclosure provides the methodof any one of Methods 46-49, wherein the one or more polynucleotideencoding one or more deoxyribonucleic acid (DNA) endonuclease isformulated into a lipid nanoparticle, and the one or more gRNA or one ormore sgRNA is delivered to the cell ex vivo by electroporation and thedonor template is delivered to the cell by an adeno-associated virus(AAV) vector.

In another method, Method 53, the present disclosure provides the methodof any one of Methods 46-49, wherein the one or more polynucleotideencoding one or more deoxyribonucleic acid (DNA) endonuclease isformulated into a liposome or lipid nanoparticle which also comprisesthe one or more gRNA or one or more sgRNA and the donor template.

The present disclosure also provides a composition, Composition 1,comprising a single-molecule guide RNA comprising: at least a spacersequence selected from SEQ ID NOs: 5305-20697.

In another composition, Composition 2, the present disclosure providesthe single-molecule guide RNA of Composition 1, wherein thesingle-molecule guide RNA further comprises a spacer extension region.

In another composition, Composition 3, the present disclosure providesthe single-molecule guide RNA of Composition 1, wherein thesingle-molecule guide RNA further comprises a tracrRNA extension region.

In another composition, Composition 4, the present disclosure providesthe single-molecule guide RNA of any one of Compositions 1-3, whereinthe single-molecule guide RNA is chemically modified.

In another composition, Composition 5, the present disclosure provides asingle-molecule guide RNA of Compositions 1-4 pre-complexed with a DNAendonuclease.

In another composition, Composition 6, the present disclosure providesthe composition of Composition 5, wherein the DNA endonuclease is a Cas9or Cpfl endonuclease.

In another composition, Composition 7, the present disclosure providesthe composition of Composition 6, wherein the Cas9 or Cpfl endonucleaseis selected from a group consisting of: S. pyogenes Cas9, S. aureusCas9, N. meningitides Cas9, S. thermophilus CRISPR1 Cas9, S.thermophilus CRISPR 3 Cas9, T. denticola Cas9, L. bacterium ND2006 Cpfland Acidaminococcus sp. BV3L6 Cpfl, and variants having at least 90%homology to the endonucleases.

In another composition, Composition 8, the present disclosure providesthe composition of Composition 7, wherein the Cas9 or Cpfl endonucleasecomprises one or more nuclear localization signals (NLSs).

In another composition, Composition 9, the present disclosure providesthe composition of Composition 8, wherein at least one NLS is at orwithin 50 amino acids of the amino-terminus of the Cas9 or Cpflendonucelase and/or at least one NLS is at or within 50 amino acids ofthe carboxy-terminus of the Cas9 or Cpfl endonucelase.

In another compostion, Composition 10, the present disclosure provides aDNA encoding the single-molecule guide RNA of any of Compositions 1-4.

In another composition, Composition 11, the present disclosure providesa non-naturally occurring CRISPR/Cas system comprising a polynucleotideencoding a Cas9 or Cpfl endonuclease and at least one single-moleculeguide RNA of Compositions 1-4.

In another composition, Composition 12, the present disclosure providesthe CRISPR/Cas system of Composition 11, wherein the polynucleotideencoding a Cas9 or Cpfl endonuclease is selected from the groupconsisting of: S. pyogenes Cas9, S. aureus Cas9, N. meningitides Cas9,S. thermophilus CRISPR1 Cas9, S. thermophilus CRISPR 3 Cas9, T.denticola Cas9, L. bacterium ND2006 Cpfl and Acidaminococcus sp. BV3L6Cpfl, and variants having at least 90% homology to the endonucleases.

In another composition, Composition 13, the present disclosure providesthe CRISPR/Cas system of Composition 12, wherein the polynucleotideencoding a Cas9 or Cpfl endonuclease comprises one or more nuclearlocalization signals (NLSs).

In another composition, Composition 14, the present disclosure providesthe CRISPR/Cas system of Composition 13, wherein at least one NLS is ator within 50 amino acids of the amino-terminus of the polynucleotideencoding a Cas9 or Cpfl endonuclease and/or at least one NLS is at orwithin 50 amino acids of the carboxy-terminus of the polynucleotideencoding a Cas9 or Cpfl endonuclease.

In another composition, Composition 15, the present disclosure providesthe CRISPR/Cas system of Composition 14, wherein the polynucleotideencoding a Cas9 or Cpfl endonuclease is codon optimized for expressionin a eukaryotic cell.

In another composition, Composition 16, the present disclosure providesa DNA encoding the CRISPR/Cas system of any one of Compositions 11-15.

In another composition, Composition 17, the present disclosure providesa vector comprising the DNA of Compositions 11 or 16.

In another composition, Composition 18, the present disclosure providesthe vector of Composition 17, wherein the vector is a plasmid.

In another composition, Composition 19, the present disclosure providesthe vector of Composition 17, wherein the vector is an AAV vectorparticle.

In another composition, Composition 20, the present disclosure providesthe vector of Composition 19, wherein the AAV vector particle isselected from the group consisting of any of those disclosed in SEQ IDNOs: 4734-5302 and Table 2.

IX. Definitions

The term “comprising” or “comprises” is used in reference tocompositions, methods, and respective component(s) thereof, that areessential to the present disclosure, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting essentially of” refers to those elements requiredfor a given aspect. The term permits the presence of additional elementsthat do not materially affect the basic and novel or functionalcharacteristic(s) of that aspect of the present disclosure.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the aspect.

The singular forms “a,” “an,” and “the” include plural references,unless the context clearly dictates otherwise.

Any numerical range recited in this specification describes allsub-ranges of the same numerical precision (i.e., having the same numberof specified digits) subsumed within the recited range. For example, arecited range of “1.0 to 10.0” describes all sub-ranges between (andincluding) the recited minimum value of 1.0 and the recited maximumvalue of 10.0, such as, for example, “2.4 to 7.6,” even if the range of“2.4 to 7.6” is not expressly recited in the text of the specification.Accordingly, the Applicant reserves the right to amend thisspecification, including the claims, to expressly recite any sub-rangeof the same numerical precision subsumed within the ranges expresslyrecited in this specification. All such ranges are inherently describedin this specification such that amending to expressly recite any suchsub-ranges will comply with written description, sufficiency ofdescription, and added matter requirements, including the requirementsunder 35 U.S.C. § 112(a) and Article 123(2) EPC. Also, unless expresslyspecified or otherwise required by context, all numerical parametersdescribed in this specification (such as those expressing values,ranges, amounts, percentages, and the like) may be read as if prefacedby the word “about,” even if the word “about” does not expressly appearbefore a number. Additionally, numerical parameters described in thisspecification should be construed in light of the number of reportedsignificant digits, numerical precision, and by applying ordinaryrounding techniques. It is also understood that numerical parametersdescribed in this specification will necessarily possess the inherentvariability characteristic of the underlying measurement techniques usedto determine the numerical value of the parameter.

The details of one or more examples of the present disclosure are setforth in the accompanying description below. Although any materials andmethods similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, the preferredmaterials and methods are now described. Other features, objects andadvantages of the present disclosure will be apparent from thedescription. In the description, the singular forms also include theplural unless the context clearly dictates otherwise. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this disclosure belongs. In the case of conflict, the presentdescription will control.

The present disclosure is further illustrated by the followingnon-limiting examples.

X. Examples

The present disclosure will be more fully understood by reference to thefollowing examples, which provide illustrative non-limiting aspects ofthe invention.

The examples describe the use of the CRISPR system as an illustrativegenome editing technique to create defined therapeutic genomicdeletions, insertions, or replacements, termed “genomic modifications”herein, in the DMPK gene that lead to permanent deletion or correctionof expanded trinucleotide repeats in the DMPK gene, knock-out of theDMPK gene, or correction of the entire gene or correction of mutationswithin the gene, that restore DMPK protein activity.

All tested gRNAs can be used for an HDR/correction based editingapproach. Single gRNAs can be used to induce insertions and deletionsdisrupting the expression of the mutant DMPK gene. Selected pairs ofgRNAs can be used to make deletions in the DMPK gene that disruptexpression and/or remove the mutant DMPK sequence. Selected pairs ofgRNAs can be used to make deletions that remove the repeat expansion.

Various Cas orthologs are evaluated for cutting. gRNAs are delivered asRNA and expressed from the U6 promoter in plasmids. The correspondingCas protein is either knocked into the cell line of interest andconstitutively expressed, delivered as mRNA, or delivered as protein.The gRNA activity in all formats is evaluated using TIDE analysis inHEK293T cells.

Introduction of the defined therapeutic modifications described aboverepresents a novel therapeutic strategy for the potential ameliorationof Myotonic Dystrophy Type 1 and related disorders, as described andillustrated herein.

Example 1 - CRISPR/SpCas9 Target Sites for the DMPK Gene

Regions of the DMPK gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence NRG.gRNA 20 bp spacer sequences corresponding to the PAM were thenidentified, as shown in SEQ ID NOs: 6368 - 15236 of the SequenceListing.

Example 2 - CRISPR/SaCas9 Target Sites for the DMPK Gene

Regions of the DMPK gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNGRRT. gRNA 20 bp spacer sequences corresponding to the PAM were thenidentified, as shown in SEQ ID NOs: 5400 -6048 of the Sequence Listing.

Example 3 - CRISPR/StCas9 Target Sites for the DMPK Gene

Regions of the DMPK gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNAGAAW. gRNA 20 bp spacer sequences corresponding to the PAM were thenidentified, as shown in SEQ ID NOs: 5333 -5399 of the Sequence Listing.

Example 4 - CRISPR/TdCas9 Target Sites for the DMPK Gene

Regions of the DMPK gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNAAAAC. gRNA 20 bp spacer sequences corresponding to the PAM were thenidentified, as shown in SEQ ID NOs: 5305 -5332 of the Sequence Listing.

Example 5 - CRISPR/NmCas9 Target Sites for the DMPK Gene

Regions of the DMPK gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequenceNNNNGATT. gRNA 20 bp spacer sequences corresponding to the PAM were thenidentified, as shown in SEQ ID NOs: 6049 -6367 of the Sequence Listing.

Example 6 - CRISPR/Cpf1 Target Sites for the DMPK Gene

Regions of the DMPK gene were scanned for target sites. Each area wasscanned for a protospacer adjacent motif (PAM) having the sequence YTN.gRNA 20 bp spacer sequences corresponding to the PAM were thenidentified, as shown in SEQ ID NOs: 15237 - 20697 of the SequenceListing.

Example 7 - Bioinformatics Analysis of the Guide Strands

Candidate guides will then screened and selected in a single process ormulti-step process that involves both theoretical binding andexperimentally assessed activity at both on-target and off-target sites.By way of illustration, candidate guides having sequences that match aparticular on-target site, such as a site within the DMPK gene, withadjacent PAM can be assessed for their potential to cleave at off-targetsites having similar sequences, using one or more of a variety ofbioinformatics tools available for assessing off-target binding, asdescribed and illustrated in more detail below, in order to assess thelikelihood of effects at chromosomal positions other than thoseintended.

Candidates predicted to have relatively lower potential for off-targetactivity can then be assessed experimentally to measure their on-targetactivity, and then off-target activities at various sites. Preferredguides have sufficiently high on-target activity to achieve desiredlevels of gene editing at the selected locus, and relatively loweroff-target activity to reduce the likelihood of alterations at otherchromosomal loci. The ratio of on-target to off-target activity is oftenreferred to as the “specificity” of a guide.

For initial screening of predicted off-target activities, there are anumber of bioinformatics tools known and publicly available that can beused to predict the most likely off-target sites; and since binding totarget sites in the CRISPR/Cas9 or CRISPR/Cpf1 nuclease system is drivenby Watson-Crick base pairing between complementary sequences, the degreeof dissimilarity (and therefore reduced potential for off-targetbinding) is essentially related to primary sequence differences:mismatches and bulges, i.e. bases that are changed to anon-complementary base, and insertions or deletions of bases in thepotential off-target site relative to the target site. An exemplarybioinformatics tool called COSMID (CRISPR Off-target Sites withMismatches, Insertions and Deletions) (available on the web atcrispr.bme.gatech.edu) compiles such similarities. Other bioinformaticstools include, but are not limited to autoCOSMID and CCTop.

Bioinformatics were used to minimize off-target cleavage in order toreduce the detrimental effects of mutations and chromosomalrearrangements. Studies on CRISPR/Cas9 systems suggested the possibilityof off-target activity due to non-specific hybridization of the guidestrand to DNA sequences with base pair mismatches and/or bulges,particularly at positions distal from the PAM region. Therefore, it isimportant to have a bioinformatics tool that can identify potentialoff-target sites that have insertions and/or deletions between the RNAguide strand and genomic sequences, in addition to base-pair mismatches.Bioinformatics tools based upon the off-target prediction algorithmCCTop were used to search genomes for potential CRISPR off-target sites(CCTop is available on the web at crispr.cos.uni-heidelberg.de/). Theoutput ranked lists of the potential off-target sites based on thenumber and location of mismatches, allowing more informed choice oftarget sites, and avoiding the use of sites with more likely off-targetcleavage.

Additional bioinformatics pipelines are employed that weigh theestimated on- and/or off-target activity of gRNA targeting sites in aregion. Other features that may be used to predict activity includeinformation about the cell type in question, DNA accessibility,chromatin state, transcription factor binding sites, transcriptionfactor binding data, and other CHIP-seq data. Additional factors areweighed that predict editing efficiency, such as relative positions anddirections of pairs of gRNAs, local sequence features andmicro-homologies.

Initial evaluation and screening of CRISPR/Cas9 target sites focused onthe 3’ untranslated region (UTR) of DMPK.

Initial bioninformatics analysis identified, 173 SpCas9 gRNAs targetingthe 3’UTR of DMPK. A prioritized list was created, which included 169 ofthe 173 SpCas9 gRNAs targeting the 3’UTR of DMPK. These gRNAs weretested for cutting efficiencies using spCas9 (Table 7).

TABLE 7 SEQ ID NO. Name Sequence 7388 DMPK 3UTR Spy T107GTGCATGACGCCCTGCTCTG 12002 DMPK 3UTR Spy T131 GCCAGACGCTCCCCAGAGCA 7372DMPK 3UTR Spy T18 TCGTCCTCCGACTCGCTGAC 7389 DMPK 3UTR Spy T83TGTGCATGACGCCCTGCTCT 12007 DMPK 3UTR Spy T14 CTTTGCGAACCAACGATAGG 11923DMPK 3UTR Spy T150 CAGAGCTTTGGGCAGATGGA 11977 DMPK 3UTR Spy T73CTCCGAGAGCAGCGCAAGTG 7386 DMPK 3UTR Spy T113 GCCCTGCTCTGGGGAGCGTC 11922DMPK 3UTR Spy T146 CCAGAGCTTTGGGCAGATGG 11953 DMPK 3UTR Spy T98AACGTGGATTGGGGTTGTTG 7393 DMPK 3UTR Spy T20 CACGCACCCCCACCTATCGT 11960DMPK 3UTR Spy T108 GTAGCCTGTCAGCGAGTCGG 12016 DMPK 3UTR Spy T79CGTGGAGGATGGAACACGGA 12006 DMPK 3UTR Spy T13 GCACTTTGCGAACCAACGAT 11965DMPK 3UTR Spy T10 AATATCCAAACCGCCGAAGC 12078 DMPK 3UTR Spy T35CGGAGCGGTTGTGAACTGGC 11932 DMPK 3UTR Spy T49 TATTCGCGAGGGTCGGGGGT 7382DMPK 3UTR Spy T66 TTTGCCAAACCCGCTTTTTC 11942 DMPK 3UTR Spy T72GGGACAGACAATAAATACCG 7367 DMPK 3UTR Spy T69 ACTGAGACCCCGACATTCCT 11982DMPK 3UTR Spy T160 GAGCAGCGCAAGTGAGGAGG 7375 DMPK 3UTR Spy T53GCCGGCTCCGCCCGCTTCGG 12001 DMPK 3UTR Spy T114 CGCCAGACGCTCCCCAGAGC 12094DMPK 3UTR Spy T55 CCGGAGTCGAAGACAGTTCT 11947 DMPK 3UTR Spy T50GTCTCAGTGCATCCAAAACG 7460 DMPK 3UTR Spy T8 TAGAACTGTCTTCGACTCCG 11944DMPK 3UTR Spy T80 AATAAATACCGAGGAATGTC 12088 DMPK 3UTR Spy T39GGGCACTCAGTCTTCCAACG 11952 DMPK 3UTR Spy T91 AAACGTGGATTGGGGTTGTT 12010DMPK 3UTR Spy T24 TGCGAACCAACGATAGGTGG 11962 DMPK 3UTR Spy T43TGTCAGCGAGTCGGAGGACG 11966 DMPK 3UTR Spy T26 ATCCAAACCGCCGAAGCGGG 12087DMPK 3UTR Spy T57 CGGGCACTCAGTCTTCCAAC 7369 DMPK 3UTR Spy T74AACAACCCCAATCCACGTTT 12008 DMPK 3UTR Spy T22 TTTGCGAACCAACGATAGGT 7384DMPK 3UTR Spy T97 CGATCTCTGCCTGCTTACTC 7363 DMPK 3UTR Spy T25CCCCGACCCTCGCGAATAAA 12095 DMPK 3UTR Spy T45 CGGAGTCGAAGACAGTTCTA 12073DMPK 3UTR Spy T103 GCTGGGCGGAGACCCACGCT 7462 DMPK 3UTR Spy T42CCTAGAACTGTCTTCGACTC 7456 DMPK 3UTR Spy T87 CCGTTGGAAGACTGAGTGCC 12086DMPK 3UTR Spy T104 CCGGGCACTCAGTCTTCCAA 7454 DMPK 3UTR Spy T58GTTGGAAGACTGAGTGCCCG 11981 DMPK 3UTR Spy T135 AGAGCAGCGCAAGTGAGGAG 12015DMPK 3UTR Spy T147 GGTGCGTGGAGGATGGAACA 12080 DMPK 3UTR Spy T134GGTTGTGAACTGGCAGGCGG 11949 DMPK 3UTR Spy T44 GTGCATCCAAAACGTGGATT 7377DMPK 3UTR Spy T122 GCTGCTCTCGGAGCCCCAGC 7376 DMPK 3UTR Spy T48CCAGCCGGCTCCGCCCGCTT 7370 DMPK 3UTR Spy T36 CCGACTCGCTGACAGGCTAC 11994DMPK 3UTR Spy T41 AGCAAATTTCCCGAGTAAGC 7448 DMPK 3UTR Spy T2GTTCACAACCGCTCCGAGCG 12071 DMPK 3UTR Spy T154 ATCACAGGACTGGAGCTGGG 11945DMPK 3UTR Spy T33 ATAAATACCGAGGAATGTCG 11958 DMPK 3UTR Spy T120CCTGTAGCCTGTCAGCGAGT 11987 DMPK 3UTR Spy T28 GCGCGGGATCCCCGAAAAAG 11988DMPK 3UTR Spy T30 CGCGGGATCCCCGAAAAAGC 11969 DMPK 3UTR Spy T121CCGAAGCGGGCGGAGCCGGC 7385 DMPK 3UTR Spy T96 GCGATCTCTGCCTGCTTACT 12075DMPK 3UTR Spy T61 GCGGAGACCCACGCTCGGAG 7461 DMPK 3UTR Spy T64CTAGAACTGTCTTCGACTCC 11964 DMPK 3UTR Spy T34 AAATATCCAAACCGCCGAAG 12070DMPK 3UTR Spy T117 CGGATCACAGGACTGGAGCT 12079 DMPK 3UTR Spy T106AGCGGTTGTGAACTGGCAGG 7455 DMPK 3UTR Spy T105 CGTTGGAAGACTGAGTGCCC 7380DMPK 3UTR Spy T93 CTCCTCACTTGCGCTGCTCT 12067 DMPK 3UTR Spy T102GCGGGCCCGGATCACAGGAC 12069 DMPK 3UTR Spy T115 CCGGATCACAGGACTGGAGC 11926DMPK 3UTR Spy T4 GGAGGGCCTTTTATTCGCGA 11950 DMPK 3UTR Spy T60TGCATCCAAAACGTGGATTG 11927 DMPK 3UTR Spy T12 GGCCTTTTATTCGCGAGGGT 11937DMPK 3UTR Spy T137 TCGGGGGTGGGGGTCCTAGG 11929 DMPK 3UTR Spy T6CCTTTTATTCGCGAGGGTCG 11979 DMPK 3UTR Spy T100 CGAGAGCAGCGCAAGTGAGG 11943DMPK 3UTR Spy T111 CAATAAATACCGAGGAATGT 12082 DMPK 3UTR Spy T143GAACTGGCAGGCGGTGGGCG 11971 DMPK 3UTR Spy T139 GAAGCGGGCGGAGCCGGCTG 11928DMPK 3UTR Spy T16 GCCTTTTATTCGCGAGGGTC 11980 DMPK 3UTR Spy T119GAGAGCAGCGCAAGTGAGGA 7424 DMPK 3UTR Spy T31 GGGTCCGCGGCCGGCGAACG 11931DMPK 3UTR Spy T54 TTATTCGCGAGGGTCGGGGG 11989 DMPK 3UTR Spy T27GATCCCCGAAAAAGCGGGTT 12061 DMPK 3UTR Spy T84 CTCCCTCCCCGGCCGCTAGG 7422DMPK 3UTR Spy T21 GGCCGGCGAACGGGGCTCGA 12051 DMPK 3UTR Spy T133CAGCAGCATTCCCGGCTACA 7361 DMPK 3UTR Spy T129 CCTCCATCTGCCCAAAGCTC 12089DMPK 3UTR Spy T99 TCAGTCTTCCAACGGGGCCC 11983 DMPK 3UTR Spy T163AGCAGCGCAAGTGAGGAGGG 7447 DMPK 3UTR Spy T11 TTCACAACCGCTCCGAGCGT 7439DMPK 3UTR Spy T81 GGGCCCGCCCCCTAGCGGCC 12053 DMPK 3UTR Spy T3ACCCTTCGAGCCCCGTTCGC 12084 DMPK 3UTR Spy T90 CGGCTTCTGTGCCGTGCCCC 12081DMPK 3UTR Spy T75 GTTGTGAACTGGCAGGCGGT 11930 DMPK 3UTR Spy T1CTTTTATTCGCGAGGGTCGG 12058 DMPK 3UTR Spy T152 CCCCTCCCTCCCCGGCCGCT 12049DMPK 3UTR Spy T149 AGCAGCAGCAGCAGCATTCC 12019 DMPK 3UTR Spy T136GCCCGGCTTGCTGCCTTCCC 7427 DMPK 3UTR Spy T156 GGAGGGGCCGGGTCCGCGGC 12060DMPK 3UTR Spy T89 CCTCCCTCCCCGGCCGCTAG 12083 DMPK 3UTR Spy T92GCGGCTTCTGTGCCGTGCCC 12059 DMPK 3UTR Spy T95 CCCTCCCTCCCCGGCCGCTA 7398DMPK 3UTR Spy T158 GGCAAACTGCAGGCCTGGGA 7404 DMPK 3UTR Spy T67GCTGAGGCCCTGACGTGGAT 7365 DMPK 3UTR Spy T130 TTTATTGTCTGTCCCCACCT 7438DMPK 3UTR Spy T86 GGCCCGCCCCCTAGCGGCCG 7432 DMPK 3UTR Spy T138CCCTAGCGGCCGGGGAGGGA 7441 DMPK 3UTR Spy T63 GATCCGGGCCCGCCCCCTAG 7426DMPK 3UTR Spy T38 CCGGGTCCGCGGCCGGCGAA 7402 DMPK 3UTR Spy T62GACGTGGATGGGCAAACTGC 7431 DMPK 3UTR Spy T164 CCTAGCGGCCGGGGAGGGAG 7443DMPK 3UTR Spy T125 CAGCTCCAGTCCTGTGATCC 7421 DMPK 3UTR Spy T17GCCGGCGAACGGGGCTCGAA 12011 DMPK 3UTR Spy T76 CAACGATAGGTGGGGGTGCG 7395DMPK 3UTR Spy T148 GGCCTGGGAAGGCAGCAAGC 12022 DMPK 3UTR Spy T29GCAGTTTGCCCATCCACGTC 12066 DMPK 3UTR Spy T82 AGGGGGCGGGCCCGGATCAC 7425DMPK 3UTR Spy T19 CGGGTCCGCGGCCGGCGAAC 12062 DMPK 3UTR Spy T88CCTCCCCGGCCGCTAGGGGG 7390 DMPK 3UTR Spy T46 TTGTGCATGACGCCCTGCTC 7381DMPK 3UTR Spy T9 TTGCCAAACCCGCTTTTTCG 7444 DMPK 3UTR Spy T112CCAGCTCCAGTCCTGTGATC 7406 DMPK 3UTR Spy T126 GCCAGGCTGAGGCCCTGACG 7440DMPK 3UTR Spy T85 CGGGCCCGCCCCCTAGCGGC 11970 DMPK 3UTR Spy T70CGAAGCGGGCGGAGCCGGCT 11968 DMPK 3UTR Spy T68 ACCGCCGAAGCGGGCGGAGC 11954DMPK 3UTR Spy T116 ACGTGGATTGGGGTTGTTGG 11951 DMPK 3UTR Spy T94AAAACGTGGATTGGGGTTGT 7405 DMPK 3UTR Spy T110 GGCTGAGGCCCTGACGTGGA 7453DMPK 3UTR Spy T118 AAGACTGAGTGCCCGGGGCA 12023 DMPK 3UTR Spy T59CAGTTTGCCCATCCACGTCA 7419 DMPK 3UTR Spy T37 GCTCGAAGGGTCCTTGTAGC 11948DMPK 3UTR Spy T52 AGTGCATCCAAAACGTGGAT 11939 DMPK 3UTR Spy T144GGGGGTGGGGGTCCTAGGTG 12013 DMPK 3UTR Spy T123 CGATAGGTGGGGGTGCGTGG 12063DMPK 3UTR Spy T71 CTCCCCGGCCGCTAGGGGGC 12056 DMPK 3UTR Spy T167GGACCCGGCCCCTCCCTCCC 7401 DMPK 3UTR Spy T132 GGATGGGCAAACTGCAGGCC 11934DMPK 3UTR Spy T159 TTCGCGAGGGTCGGGGGTGG 7428 DMPK 3UTR Spy T157GGAGGGAGGGGCCGGGTCCG 12028 DMPK 3UTR Spy T153 GCCTGGCCGAAAGAAAGAAA 12055DMPK 3UTR Spy T51 CCGTTCGCCGGCCGCGGACC 12025 DMPK 3UTR Spy T128TCCACGTCAGGGCCTCAGCC 12014 DMPK 3UTR Spy T162 AGGTGGGGGTGCGTGGAGGA 12054DMPK 3UTR Spy T40 CGAGCCCCGTTCGCCGGCCG 7435 DMPK 3UTR Spy T77CGCCCCCTAGCGGCCGGGGA 11985 DMPK 3UTR Spy T145 GCAAGTGAGGAGGGGGGCGC 7409DMPK 3UTR Spy T109 ACCATTTCTTTCTTTCGGCC 7418 DMPK 3UTR Spy T56CTCGAAGGGTCCTTGTAGCC 7433 DMPK 3UTR Spy T140 CCCCTAGCGGCCGGGGAGGG 11933DMPK 3UTR Spy T101 ATTCGCGAGGGTCGGGGGTG 7407 DMPK 3UTR Spy T168TCTTTCTTTCGGCCAGGCTG 7436 DMPK 3UTR Spy T78 CCGCCCCCTAGCGGCCGGGG 11925DMPK 3UTR Spy T5 TGGAGGGCCTTTTATTCGCG 7400 DMPK 3UTR Spy T141GATGGGCAAACTGCAGGCCT 7459 DMPK 3UTR Spy T32 CTTCGACTCCGGGGCCCCGT 11938DMPK 3UTR Spy T124 CGGGGGTGGGGGTCCTAGGT 7374 DMPK 3UTR Spy T7CTCCGCCCGCTTCGGCGGTT 7430 DMPK 3UTR Spy T172 GCGGCCGGGGAGGGAGGGGC 12009DMPK 3UTR Spy T15 TTGCGAACCAACGATAGGTG 7394 DMPK 3UTR Spy T155GCCTGGGAAGGCAGCAAGCC 7383 DMPK 3UTR Spy T65 TTTTGCCAAACCCGCTTTTT 7411DMPK 3UTR Spy T142 CACAGACCATTTCTTTCTTT 12076 DMPK 3UTR Spy T23CGCTCGGAGCGGTTGTGAAC 7429 DMPK 3UTR Spy T171 CGGCCGGGGAGGGAGGGGCC 12017DMPK 3UTR Spy T47 AGGATGGAACACGGACGGCC 12064 DMPK 3UTR Spy T127CGGCCGCTAGGGGGCGGGCC 11936 DMPK 3UTR Spy T161 GGGTCGGGGGTGGGGGTCCT 11984DMPK 3UTR Spy T151 CGCAAGTGAGGAGGGGGGCG 7413 DMPK 3UTR Spy T165CTGCTGCTGCTGCTGCTGGG

Note that the SEQ ID NOs represent the DNA sequence of the genomictarget, while the gRNA or sgRNA spacer sequence will be the RNA versionof the DNA sequence.

Initial bioninformatics analysis identified 28 SaCas9 gRNAs targetingthe 3’UTR of DMPK. A prioritized list was created, which included 28 ofthe 28 SaCas9 gRNAs targeting the 3’UTR of DMPK. These gRNAs were testedfor cutting efficiencies using SaCas9 (Table 8).

TABLE 8 SEQ ID NO. Name Sequence 5446 DMPK_3UTR_Sau_T5CGGCCGGCGAACGGGGCTCG 5448 DMPK_3UTR_Sau_T8 AGTTCACAACCGCTCCGAGC 5449DMPK_3UTR_Sau_T18 TCCGGGGCCCCGTTGGAAGA 5808 DMPK_3UTR_Sau_T6CCCGGAGTCGAAGACAGTTC 5798 DMPK_3UTR_Sau_T15 AGTGCATCCAAAACGTGGAT 5807DMPK_3UTR_Sau_T1 TCAGTCTTCCAACGGGGCCC 5794 DMPK_3UTR_Sau_T9TATTCGCGAGGGTCGGGGGT 5792 DMPK_3UTR_Sau_T2 ATGGAGGGCCTTTTATTCGC 5796DMPK_3UTR_Sau_T25 CAATAAATACCGAGGAATGT 5800 DMPK_3UTR_Sau_T19GGGGGTCCTGTAGCCTGTCA 5443 DMPK_3UTR_Sau_T23 GGCCAGGCTGAGGCCCTGAC 5439DMPK_3UTR_Sau_T26 GACCCCCACCCCCGACCCTC 5799 DMPK_3UTR_Sau_T22AAACGTGGATTGGGGTTGTT 5803 DMPK_3UTR_Sau_T24 GTTTGGCAAAAGCAAATTTC 5804DMPK_3UTR_Sau_T11 TTTGCGAACCAACGATAGGT 5802 DMPK_3UTR_Sau_T7GGCGCGGGATCCCCGAAAAA 5440 DMPK_3UTR_Sau_T10 CAACAACCCCAATCCACGTT 5444DMPK_3UTR_Sau_T28 GCTGCTGCTGCTGCTGCTGG 5447 DMPK_3UTR_Sau_T27AGCGGCCGGGGAGGGAGGGG 5806 DMPK_3UTR_Sau_T16 CCGGCCGCTAGGGGGCGGGC 5442DMPK_3UTR_Sau_T21 TTTGCCAAACCCGCTTTTTC 5793 DMPK_3UTR_Sau_T3GCCTTTTATTCGCGAGGGTC 5441 DMPK_3UTR_Sau_T4 GCTCCGCCCGCTTCGGCGGT 5797DMPK_3UTR_Sau_T17 GGTCTCAGTGCATCCAAAAC 5795 DMPK_3UTR_Sau_T12GGGACAGACAATAAATACCG 5801 DMPK_3UTR_Sau_T20 CGCAAGTGAGGAGGGGGGCG 5805DMPK_3UTR_Sau_T13 ACGATAGGTGGGGGTGCGTG 5445 DMPK_3UTR_Sau_T14CTCGAAGGGTCCTTGTAGCC

Pairs of gRNAs that cut on either side of the expanded repeat sequencecan be used to excise the repeats. Single gRNAs that cut proximal torepeats can be used to partially or fully truncate the repeat sequences.Pairs of gRNAs on either side of the repeats can be used for excisingthe repeats and replacing the repeats with wildtype sequence.

Two SpCas9 gRNAs and one SaCas9 gRNA that overlap with the repeat have aperfect off target elsewhere in the genome, so may not be useful for anediting based strategy.

Example 8 - Testing of Preferred Guides in in Vitro Transcribed (IVT)gRNA Screen

To identify a large spectrum of pairs of gRNAs able to edit the cognateDNA target region, an in vitro transcribed (IVT) gRNA screen wasconducted. The relevant genomic sequence was submitted for analysisusing a gRNA design software. The resulting list of gRNAs was narrowedto a select list of gRNAs as described above based on uniqueness ofsequence (only gRNAs without a perfect match somewhere else in thegenome were screened) and minimal predicted off targets. This set ofgRNAs was in vitro transcribed, and transfected using LipofectamineMessengerMAX into HEK293T cells that constitutively express Cas9. Cellswere harvested 48 hours post transfection, the genomic DNA was isolated,and cutting efficiency was evaluated using TIDE analysis. (FIGS. 2A-G;FIGS. 3A-C; FIGS. 4A-B; and FIG. 5 ).

The gRNA or pairs of gRNA with significant activity can then be followedup in cultured cells to measure correction of the DMPK mutation.Off-target events can be followed again. A variety of cells can betransfected and the level of gene correction and possible off-targetevents measured. These experiments allow optimization of nuclease anddonor design and delivery.

Example 9 - Testing of Preferred Guides in Cells for Off-Target Activity

The gRNAs having the best on-target activity from the IVT screen in theabove example are tested for off-target activity using Hybrid captureassays, GUIDE Seq. and whole genome sequencing in addition to othermethods.

Example 10 - Testing Different Approaches for HDR Gene Editing

After testing the gRNAs for both on-target activity and off-targetactivity, repeat expansion correction and whole gene correctionstrategies will be tested for HDR gene editing.

For the whole gene correction approach, a single-stranded ordouble-stranded DNA having homologous arms to the DMPK chromosomalregion may include more than 40 nt of the first exon (the first codingexon) of the DMPK gene, the complete CDS of the DMPK gene and 3’ UTR ofthe DMPK gene, and at least 40 nt of the following intron. Thesingle-stranded or double-stranded DNA having homologous arms to theDMPK chromosomal region may include more than 80 nt of the first exon ofthe DMPK gene, the complete CDS of the DMPK gene and 3’ UTR of the DMPKgene, and at least 80 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the DMPK chromosomalregion may include more than 100 nt of the first exon of the DMPK gene,the complete CDS of the DMPK gene and 3’ UTR of the DMPK gene, and atleast 100 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the DMPK chromosomalregion may include more than 150 nt of the first exon of the DMPK gene,the complete CDS of the DMPK gene and 3’ UTR of the DMPK gene, and atleast 150 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the DMPK chromosomalregion may include more than 300 nt of the first exon of the DMPK gene,the complete CDS of the DMPK gene and 3’ UTR of the DMPK gene, and atleast 300 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the DMPK chromosomalregion may include more than 400 nt of the first exon of the DMPK gene,the complete CDS of the DMPK gene and 3’ UTR of the DMPK gene, and atleast 400 nt of the following intron.

Alternatively, the DNA template will be delivered by a recombinant AAVparticle such as those taught herein.

A knock-in of DMPK cDNA can be performed into any selected chromosomallocation, including the DMPK gene locus or in a “safe-harbor” locus,i.e., AAVS1 (PPP1R12C), ALB, Angptl3, ApoC3, ASGR2, CCR5, FIX (F9),Gys2, HGD, Lp(a), Pcsk9, Serpinal, TF, and/or TTR. Assessment ofefficiency of HDR mediated knock-in of cDNA into the first exon canutilize cDNA knock-in into “safe harbor” sites such as: single-strandedor double-stranded DNA having homologous arms to one of the followingregions, for example: exon 1-2 of AAVS1 (PPP1R12C), exon 1-2 of ALB,exon 1-2 of Angptl3, exon 1-2 of ApoC3, exon 1-2 of ASGR2, exon 1-2 ofCCR5, exon 1-2 of FIX (F9), exon 1-2 of Gys2, exon 1-2 of HGD, exon 1-2of Lp(a), exon 1-2 of Pcsk9, exon 1-2 of Serpinal, exon 1-2 of TF, orexon 1-2 of TTR; 5’UTR correspondent to DMPK or alternative 5’ UTR,complete CDS of DMPK and 3’ UTR of DMPK or modified 3’ UTR and at least80 nt of the first intron, alternatively same DNA template sequence willbe delivered by AAV.

Example 11 - Re-Assessment of Lead CRISPR-Cas9/DNA Donor Combinations

After testing the different strategies for gene editing, the leadCRISPR-Cas9/DNA donor combinations will be re-assessed in cells forefficiency of deletion, recombination, and off-target specificity. Cas9mRNA or RNP will be formulated into lipid nanoparticles for delivery,sgRNAs will be formulated into nanoparticles or delivered as arecombinant AAV particle, and donor DNA will be formulated intonanoparticles or delivered as recombinant AAV particle.

Example 12 - In Vivo Testing in Relevant Animal Model

After the CRISPR-Cas9/DNA donor combinations have been re-assessed, thelead formulations will be tested in vivo in an animal model.

Culture in human cells allows direct testing on the human target and thebackground human genome, as described above.

Preclinical efficacy and safety evaluations can be observed throughengraftment of modified mouse or human myocytes or neurons in a mousemodel. The modified cells can be observed in the months afterengraftment.

XI. Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificexamples in accordance with the invention described herein. The scope ofthe present disclosure is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

Claims or descriptions that include “or” between one or more members ofa group are considered satisfied if one, more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process unless indicated to the contrary or otherwiseevident from the context. The present disclosure includes examples inwhich exactly one member of the group is present in, employed in, orotherwise relevant to a given product or process. The present disclosureincludes examples in which more than one, or the entire group membersare present in, employed in, or otherwise relevant to a given product orprocess.

In addition, it is to be understood that any particular example of thepresent disclosure that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such examples aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular example of the compositions of the disclosure (e.g., anyantibiotic, therapeutic or active ingredient; any method of production;any method of use; etc.) can be excluded from any one or more claims,for any reason, whether or not related to the existence of prior art.

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the present disclosure in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described examples, it isnot intended that it should be limited to any such particulars orexamples or any particular example, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

Note Regarding Illustrative Examples

While the present disclosure provides descriptions of various specificaspects for the purpose of illustrating various aspects of the presentdisclosure and/or its potential applications, it is understood thatvariations and modifications will occur to those skilled in the art.Accordingly, the invention or inventions described herein should beunderstood to be at least as broad as they are claimed, and not as morenarrowly defined by particular illustrative aspects provided herein.

Any patent, publication, or other disclosure material identified hereinis incorporated by reference into this specification in its entiretyunless otherwise indicated, but only to the extent that the incorporatedmaterial does not conflict with existing descriptions, definitions,statements, or other disclosure material expressly set forth in thisspecification. As such, and to the extent necessary, the expressdisclosure as set forth in this specification supersedes any conflictingmaterial incorporated by reference. Any material, or portion thereof,that is said to be incorporated by reference into this specification,but which conflicts with existing definitions, statements, or otherdisclosure material set forth herein, is only incorporated to the extentthat no conflict arises between that incorporated material and theexisting disclosure material. Applicants reserve the right to amend thisspecification to expressly recite any subject matter, or portionthereof, incorporated by reference herein.

1-53. (canceled)
 54. A single-molecule guide RNA comprising: a spacersequence that is an RNA sequence corresponding to any one of SEQ ID NOs:5446, 5448, 5449, 5808, 5798, 5807, 5794, 5792, 5796, 5800, 5443, 5439,5799, 5803, 5440, and
 5444. 55. The single-molecule guide RNA of claim54, wherein the single-molecule guide RNA further comprises a spacerextension region.
 56. The single-molecule guide RNA of claim 54, whereinthe single-molecule guide RNA further comprises a tracrRNA extensionregion.
 57. The single-molecule guide RNA of claim 54, wherein thesingle-molecule guide RNA is chemically modified.
 58. Thesingle-molecule guide RNA of claim 54 pre-complexed with a DNAendonuclease.
 59. The single-molecule guide RNA of claim 58, wherein theDNA endonuclease is a Cas9 or Cpfl endonuclease.
 60. The single-moleculeguide RNA of claim 59, wherein the Cas9 or Cpfl endonuclease is selectedfrom the group consisting of: S. pyogenes Cas9, S. aureus Cas9, N.meningitides Cas9, S. thermophilus CRISPR1 Cas9, S. thermophilus CRISPR3 Cas9, T. denticola Cas9, L. bacterium ND2006 Cpfl and Acidaminococcussp. BV3L6 Cpfl, and variants having at least 90% homology to saidendonucleases.
 61. The single-molecule guide RNA of claim 60, whereinthe Cas9 or Cpfl endonuclease comprises one or more nuclear localizationsignals (NLSs).
 62. The single-molecule guide RNA of claim 61, whereinat least one of the one or more NLSs is at or within 50 amino acids ofthe amino-terminus of the Cas9 or Cpfl endonuclease and/or at least oneof the one or more NLSs is at or within 50 amino acids of thecarboxy-terminus of the Cas9 or Cpfl endonuclease.
 63. A DNA encodingthe single-molecule guide RNA of claim 54 .
 64. A method for editing aDystrophia Myotonica-Protein Kinase (DMPK) gene in a cell by genomeediting comprising: introducing into the cell one or moredeoxyribonucleic acid (DNA) endonuclease or one or more polynucleotideencoding the one or more DNA endonuclease; and one or moresingle-molecule guide RNA comprising a spacer sequence that is an RNAsequence corresponding to any one of SEQ IDNOs: 5446, 5448, 5449, 5808,5798, 5807, 5794, 5792, 5796, 5800, 5443, 5439, 5799, 5803, 5440, and5444, to effect one or more single-strand breaks (SSBs) or double-strandbreaks (DSBs) within or near the DMPK gene or DMPK regulatory elementsthat results in one or more permanent insertion, deletion, or mutationof at least one nucleotide within or near the DMPK gene, therebyreducing or eliminating the expression or function of aberrant DMPK geneproducts.
 65. The method of claim 64, wherein the cell is a muscle cell.66. The method of claim 65, wherein the muscle cell is a skeletal musclecell, a smooth muscle cell, or a cardiac muscle cell.
 67. The method ofclaim 64, wherein the one or more polynucleotide encoding the one ormore DNA endonuclease is codon optimized.
 68. The method of claim 64,wherein the one or more polynucleotide encoding the one or more DNAendonuclease is chemically modified.
 69. The method of claim 64, whereinthe one or more DNA endonuclease is a Cas9 or Cpfl endonuclease.
 70. Themethod of claim 64, wherein the one or more DNA endonuclease is encodedin an AAV vector particle which also encodes the one or moresingle-molecule guide RNA.
 71. The method of claim 64, wherein the oneor more DNA endonuclease is formulated in a liposome or lipidnanoparticle which also comprises the one or more single-molecule guideRNA.
 72. A kit comprising a single-molecule guide RNA comprising aspacer sequence that is an RNA sequence corresponding to any one of SEQID NOs: 5446, 5448, 5449, 5808, 5798, 5807, 5794, 5792, 5796, 5800,5443, 5439, 5799, 5803, 5440, and 5444, or a polynucleotide encoding thesingle-molecule guide RNA.
 73. The kit of claim 72, further comprising:(a) a site-directed polypeptide; (b) a buffer, a control reagent, acontrol vector, a control RNA polynucleotide, a reagent for in vitroproduction of the polypeptide from DNA, adaptors for sequencing, or anycombination thereof; (c) instructions for using the components of thekit; or (d) any combination of (a)-(c).